Potentiation of anticancer agents

ABSTRACT

The present invention relates to carriers, conjugate and pharmaceutical compositions and their use to increase the potency of drugs and to modify the pharmacokinetics of compounds. More particularly, the present invention relates to conjugates comprising the carrier described herein and their use in the treatment and diagnostic of cancer.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to carriers conjugates and pharmaceuticalcompositions and their use to increase the potency of drugs and tomodify the pharmacokinetics of compounds. More particularly, the presentinvention relates to conjugates comprising the carrier described hereinand their use in the treatment and diagnostic of cancer.

BACKGROUND OF THE INVENTION

Clinical progress in the treatment of primary tumors has been slow andone of the problems associated with these tumors is their weak responseto anticancer drugs. The effectiveness of chemotherapy and immunotherapyhave been impaired by inherent or acquired multiple drug resistance(MDR) phenotype by cancer cells. One mechanism involved in MDR phenotypeis caused by the expression of P-glycoprotein (P-gp), a membranetransporter that pumps out various anticancer drugs from MDR1 expressingcells. P-gp is also expressed in a large number of normal secretorytissues such as kidney, liver and intestine. This efflux pump isstrongly expressed in the brain capillaries where its expression wasmainly localized in the luminal membrane of endothelial cells liningthese. In human, P-gp is encoded by two MDR genes; MDR1 and MDR3. P-gpencoded by the human MDR1 gene confers the resistance phenotype whereasP-gp encoded by the human MDR3 gene does not. Thus, P-gp may be seen asa guardian that limits the entry of drugs by expulsing them out of thebrain or out of cancer cells preventing them from reaching cytotoxicconcentrations.

Cancer cells forming brain metastases originate mostly from lung orbreast cancers, colorectal carcinoma, melanoma and urinary organ tumors.These metastases, which often occur after surgery, primary chemotherapytreatment or radiotherapy, are chemo-resistant. Chemotherapy againstbrain metastases could be effective only if it was effective for theircorresponding originate tumors. For example, it was shown that brainmetastases originating from small cell lung carcinomas and germ cellsrespond with similar rates than metastases at other sites.

Drug resistance may be an intrinsic property of tumor cells or may beacquired after treatment. The presence of the P-gp efflux pump encodedby MDR1 (also herein referred as P-glycoprotein, MDR1 P-gp or MDR1) hasbeen reported in most of the primary brain tumors where most gliomas andmore particularly endothelial cells of newly formed capillaries werestained positive for MDR1 P-gp. Thus, various studies support the ideathat the multiple drug resistance phenotype may be caused not only bythe expression of P-gp in cancer cells but also from its expression inthe newly formed endothelial cells in the tumors. MDR1 levels were alsofound significantly lower in brain metastasis from melanomas and lungadenocarcinomas. In addition, it was shown that treatments prior tosurgery have no major impact on MDR1 levels in brain metastasis frommelanomas since they were identical in patients that receivedradiotherapy, chemotherapy or both treatments. In lung metastasis, MDR1was only detected in patients that received chemotherapy indicating thatthese previous treatments may have induced its expression resulting inan acquired MDR phenotype. The lack of MDR1 expression in primary lungtumors and in their corresponding brain metastasis indicates also thatthese metastases did not acquire the same levels of P-gp expressionduring their development than the ones found in normal brain tissue.These results also indicate that the MDR1 levels of endothelial cellsfrom capillaries in brain metastasis differed from the one of primarybrain tumors. The lack of MDR1 expression in some brain metastasis mayexplain in part why some of them are more sensitive to chemotherapeuticdrugs than primary brain tumors.

Methods for transporting a compound across the blood-brain barrier havebeen described in international application no. PCT/CA2004/000011published on Jul. 22, 2004 under publication No. WO2004060403, theentire content of which is incorporated herein by reference. Briefly, inthis document, aprotinin, aprotinin fragments and analogs were presentedas a drug delivery system for the central nervous system (CNS) and fortreating CNS related diseases.

There remains a need for increasing the potency of anticancer drugs.

The present invention seeks to meet these and other needs.

SUMMARY OF THE INVENTION

The present invention relates in one aspect thereof, to a carriercomprising an amino acid sequence selected from the group consisting ofthe amino acid sequence of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2 and biologically active analogs,derivatives or fragments thereof. The aprotinin sequence as well as someexemplary embodiments of biologically active analogs may be found forexample in international application no. PCT/CA2004/000011.

The present invention also relates to a carrier consisting of an aminoacid sequence selected from the group consisting of the amino acidsequence of aprotinin, a biologically active aprotinin fragment,Angiopep-1, Angiopep-2 and biologically active analogs, derivatives orfragments thereof.

Exemplary embodiment of carriers encompassed by the present inventionincludes those which may be selected, for example, from the groupconsisting of

-   -   aprotinin (SEQ ID NO.:98),    -   an aprotinin analogue    -   an aprotinin fragment which may comprise (or may consist        essentially of) the amino acid sequence defined in SEQ ID NO.:1,    -   a biologically active analogue of SEQ ID NO.:1,    -   a biologically active fragment of SEQ ID NO.:1, and;    -   a biologically active fragment of a SEQ ID NO.:1 analogue.

More particularly, the carrier may be selected, for example, from thegroup of;

-   -   an aprotinin fragment which may comprise the amino acid sequence        defined in SEQ ID NO.:1,    -   a biologically active analogue of SEQ ID NO.:1,    -   a biologically active fragment of SEQ ID NO.:1 and;    -   a biologically active fragment of a SEQ ID NO.:1 analogue.

In accordance with the present invention the aprotinin fragment mayconsist of the sequence defined in SEQ ID NO.:1. Further in accordancewith the present invention, the aprotinin fragment may comprise SEQ IDNO.1 and may have a length of from about 19 amino acids to about 54amino acids, e.g., from 10 to 50 amino acids in length, from 10 to 30amino acids in length etc.

In accordance with the present invention, the biologically activeanalogue of SEQ ID NO.:1, may have a length of from about 19 amino acidsto about 54 amino acids (e.g., including for example 21 to 23, 25 to 34,36 to 50 and 52 to 54), or of from about 19 amino acids to about 50amino acids, or from about 19 amino acids to about 34 amino acids (e.g.,19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), of fromabout 19 amino acids to about 23 amino acids or of about 19, 20, 21, 22,23, 24, 35, 51, amino acids.

A biologically active fragment of a polypeptide (e.g., of 19 aminoacids) described herein may include for example a polypeptide of fromabout 7, 8, 9 or 10 to 18 amino acids (i.e., 11-18, 12-18, 13-18, 14-18,15-18, 16-18, 17-18). Therefore, in accordance with the presentinvention, a biologically active fragment of SEQ ID NO.:1 or of a SEQ IDNO.:1 analogue may have a length of from about 7 to about 18 amino acidsor from about 10 about 18 amino acids.

The polypeptides of the present invention may be amidated, i.e., mayhave an amidated amino acid sequence. The polypeptides of the presentinvention may be acylated.

Exemplary embodiments of the peptides of the present invention are thosehaving a Lysine at position 10 (with respect to amino acid sequence ofSEQ ID NO.:1). Other exemplary embodiments of the peptides of thepresent invention are those having a Lysine at position 15 (with respectto amino acid sequence of SEQ ID NO.:1). Further exemplary embodiment ofpeptides of the present invention are those having Lysines at positions10 and 15. The peptides of the present invention may also have a serineor cysteine at position 7 (with respect to amino acid sequence of SEQ IDNO.:1). When multimerization of peptides is desired, the peptide maypreferably comprises a cysteine at position 7. However, whenmultimerization of peptides is not required, the peptide may preferablyhave a serine at position 7.

U.S. Pat. No. 5,807,980 describes a polypeptide which is identifiedherein as SEQ ID NO.:102.

U.S. Pat. No. 5,780,265 describes a polypeptide which is identifiedherein as SEQ ID NO.:103.

The aprotinin amino acid sequence (SEQ ID NO.:98), the Angiopep-1 aminoacid sequence (SEQ ID NO.:67), as well as some sequences of biologicallyactive analogs may be found for example in international application no.PCT/CA2004/000011 published on Jul. 22, 2004 in under internationalpublication no. WO2004/060403. Additionally, international publicationNo. WO04/060403 describes a polypeptide which is identified herein asSEQ ID NO.: 104.

U.S. Pat. No. 5,118,668 describes polypeptides which has the sequenceillustrated in SEQ ID NO.: 105.

Even more particularly, the carrier may be selected, for example, fromthe group of;

-   -   a SEQ ID NO.:1 analogue which may comprise at least 35% identity        with the amino acid sequence of SEQ ID NO.:1,    -   a SEQ ID NO.:1 analogue which may comprise at least 40% identity        with the amino acid sequence of SEQ ID NO.:1,    -   a SEQ ID NO.:1 analogue which may comprise at least 50% identity        with the amino acid sequence of SEQ ID NO.:1,    -   a SEQ ID NO.:1 analogue which may comprise at least 60% identity        with the amino acid sequence of SEQ ID NO.:1,    -   a SEQ ID NO.:1 analogue which may comprise at least 70% identity        with the amino acid sequence of SEQ ID NO.:1,    -   a SEQ ID NO.:1 analogue which may comprise at least 80% identity        with the amino acid sequence of SEQ ID NO.:1,    -   a SEQ ID NO.:1 analogue which may comprise at least 90% identity        with the amino acid sequence of SEQ ID NO.:1 and;    -   a SEQ ID NO.:1 analogue which may comprise at least 95% (i.e.,        96%, 97%, 98%, 99% and 100%) identity with the amino acid        sequence of SEQ ID NO.:1.

For example, the biologically active analogue of SEQ ID NO.:1 maycomprise an amino acid sequence selected from the group consisting of anamino acid sequence defined in any one of SEQ ID NO.:2 to SEQ ID NO.:62, SEQ ID NO.: 68 to SEQ ID NO.: 93, and SEQ ID NO.:97 as well as 99,100, 101 or any of SEQ ID NO.:107-112.

Further in accordance with the present invention, the biologicallyactive analogue of SEQ ID NO.:1 may comprise the amino acid sequencedefined in SEQ ID NO.:67. This sequence may more particularly beamidated.

For example and without limitation, conjugates comprising peptides SEQID NO.: 102, 103, 104 and 105 are also encompassed by the presentinvention.

Further in accordance with the present invention, the biologicallyactive fragment of SEQ ID NO.:1 or the biologically active fragment of aSEQ ID NO.:1 analogue may comprise at least 9 or at least 10(consecutive or contiguous) amino acids of SEQ ID NO.1 or of the SEQ IDNO.:1 analogue.

The polypeptides of the present invention may have an amino acidsequence which may comprise of from between 1 to 12 amino acidsubstitutions (i.e., SEQ ID NO.:91). For example, the amino acidsubstitution may be from between 1 to 10 amino acid substitutions, orfrom 1 to 5 amino acid substitutions. In accordance with the presentinvention, the amino acid substitution may be a non-conservative aminoacid substitution or a conservative amino acid substitution.

For example, when a polypeptide of the present invention comprises aminoacids which are identical to those of SEQ ID NO.:1 and other amino acidswhich are not identical (non-identical), those which are non-identicalmay be a conservative amino acid substitution. The comparison ofidentical and non-identical amino acids may be performed by looking at acorresponding location.

Examples of SEQ ID NO.:1 analogue which may have at least 35% identityincludes for example, a polypeptide comprising (consisting of) the aminoacid sequence defined in SEQ ID NO.:91 (about 36.8% identity, i.e., 7amino acid out of 19 amino acids of SEQ ID NO.:91 are identical to SEQID NO.:1), a polypeptide comprising (consisting of) the amino acidsequence defined in SEQ ID NO.:98 (about 68.4% identity, i.e., 13 aminoacid out of 19 amino acids are identical to SEQ ID NO.:1), a polypeptidecomprising (consisting of) the amino acid sequence defined in SEQ IDNO.:67 (about 73.7% identity, i.e., 14 amino acid out of 19 amino acidsare identical to SEQ ID NO.:1), a polypeptide comprising (consisting of)the amino acid sequence defined in SEQ ID NO.: 76 (about 73.7% identity,i.e., 14 amino acid out of 19 amino acids are identical to SEQ ID NO.:1)and a polypeptide comprising (consisting of) the amino acid sequencedefined in SEQ ID NO.:5 (about 79% identity, i.e., 15 amino acid out of19 amino acids are identical to SEQ ID NO.:1).

Examples of SEQ ID NO.:1 analogue which may have at least 60% identityincludes for example, a polypeptide comprising (consisting of) the aminoacid sequence defined in SEQ ID NO.:98 (about 68.4% identity, i.e., 13amino acid out of 19 amino acids are identical to SEQ ID NO.:1), apolypeptide comprising (consisting of) the amino acid sequence definedin SEQ ID NO.:67 (about 73.7% identity, i.e., 14 amino acid out of 19amino acids are identical to SEQ ID NO.:1), a polypeptide comprising(consisting of) the amino acid sequence defined in SEQ ID NO.: 76 (about73.7% identity, i.e., 14 amino acid out of 19 amino acids are identicalto SEQ ID NO.:1) and a polypeptide comprising (consisting of) the aminoacid sequence defined in SEQ ID NO.:5 (about 79% identity, i.e., 15amino acid out of 19 amino acids are identical to SEQ ID NO.:1).

Examples of SEQ ID NO.:1 analogue which may have at least 70% identityincludes for example, a polypeptide comprising (consisting of) the aminoacid sequence defined in SEQ ID NO.:67 (about 73.7% identity, i.e., 14amino acid out of 19 amino acids are identical to SEQ ID NO.:1), SEQ IDNO.: 76 (about 73.7% identity, i.e., 14 amino acid out of 19 amino acidsare identical to SEQ ID NO.:1), SEQ ID NO.:5 (about 79% identity, i.e.,15 amino acid out of 19 amino acids are identical to SEQ ID NO.:1).

In accordance, with the present invention, the carrier may moreparticularly be selected from the group consisting of peptide Nos. 5,67, 76, 91 and peptide 97 (i.e., SEQ ID NO.:5, 67, 76, 91 and 97(Angiopep-2)).

The present invention particularly relates to the use of a carrier orthe pharmaceutical composition described herein for modifying and/orimproving the (in vivo) pharmacokinetics of a compound.

In accordance with the present invention, the compound may be selected,for example, from the group consisting of a label, a protein, a peptideand a small molecule drug and combination thereof.

Also in accordance with the present invention, the small molecule drugmay be, for example, an anticancer drug.

In accordance with the present invention the anticancer drug may beconjugated with the carrier thereby forming a conjugate. In an exemplaryembodiment of the invention, the conjugate may comprise, for example, atleast one anticancer drug molecule for each carrier molecule. In anotherexemplary embodiment of the invention, the conjugate may comprise, forexample, at least two anticancer drug molecules for each carriermolecule. In yet another exemplary embodiment of the invention, theconjugate may comprise, for example, at least three anticancer drugmolecules for each carrier molecule.

In accordance with the present invention the carrier may promoteaccumulation of the drug in a tissue such as, for example, a kidney(kidney tissue), a liver (liver tissue), an eye (eye tissue) and thelungs (lung tissue) of an individual.

Also in accordance with the present invention, the carrier may modify orimprove the bioavailability of the compound.

Further in accordance with the present invention, the carrier may alsochange the (usual) tissue distribution of the compound.

In accordance with the present invention the carrier may also promoteaccumulation of the drug in the brain (brain tissue) of an individual.

In accordance with the present invention, the brain may be a tumoralbrain.

Further in accordance with the present invention, the brain may comprisea lung cancer cell.

Also in accordance with the present invention, the carrier may promoteaccumulation of the drug in a cancer cell (e.g., intracellularaccumulation of the drug in the cancer cell).

As used herein the term “tumoral brain” refers to a brain whichcomprises a tumor, either a primary tumor or a metastasis of a differenttissue origin, such as, without limitation, a metastasis originatingfrom a lung tumor, a breast tumor, from a melanoma, from a colorectaltumor, from a tumor of an urinary organ or else. Examples of tumoralbrain cells thus include, for example, glioblastomas, and metastaticcell originating, for example, from the lung, breast, colon, urinarytract or from melanoma.

In accordance with the present invention, the carrier may thus be used,for example, for reducing the dose of a drug, necessary to achieve thesame therapeutic effect (e.g., to achieve a reduction in tumor cellgrowth, etc.).

The present invention further relates to the use of a carrier selectedfrom the group consisting of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2, biologically active analogs,derivatives or fragments and combination thereof for transporting acompound to a desired target site, a desired target tissue or a desiredtarget cell.

Examples of a small molecule drug which may be conjugated with thecarrier of the present invention and which are encompassed herewith,includes for example and without limitation, Taxol, a Taxol derivative,vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide,Taxotere, melphalan, chlorambucil, pharmaceutically acceptable salts,etc. and combination thereof as well as any drug which may be a P-gpsubstrate.

Other small molecule drug encompassed by the present invention mayinclude, for example, a drug having a group allowing it's conjugation tothe carrier of the present invention.

In accordance with the present invention, exemplary embodiments of Taxolderivatives (or analogues) include for example, derivatives disclosedand referred to in U.S. Pat. No. 6,911,549 issued on Jun. 28, 2005, theentire contents of which is incorporated herein by reference.

Examples of labels which may be conjugated with the carrier of thepresent invention and which are encompassed herewith include, forexample and without limitation, an isotope, a fluorescent label (e.g.,rhodamine), a reporter molecule (e.g., biotin), etc.

Examples of protein which may be conjugated with the carrier of thepresent invention and which are encompassed herewith includes, withoutlimitation, an antibody, an antibody fragment, a peptidic- orprotein-based drug (e.g., a positive pharmacological modulator (agonist)or an pharmacological inhibitor (antagonist)) etc.

The present invention also provides the use of a carrier selected fromthe group consisting of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2, biologically active analogs,derivatives or fragments and combination thereof for increasing thepotency of a drug. More particularly, the carrier may be used toincrease, for example, the potency of a drug which may be a P-gpsubstrate or drugs which are expulsed (i.e., expelled, ejected from acell, etc.) by P-gp or P-gp related protein (e.g., a P-gp human ormammalian allelic variant, e.g., a mdr1a and/or mdr1b isoforms from arodent, etc.).

The carrier may be used for reducing the toxicity of a drug, upon theirconjugation, and therefore allows administration of the drug at a dosewhich is higher than the recommended dose for the drug alone.

Therefore the present invention provides a method of treating a patienthaving a cancer, the method may comprise administering a conjugatecomprising the carrier of the present invention and the desired drug ata dose higher than the therapeutic dose of the drug. At comparabledosage the conjugate may be associated with less toxicity than theunconjugated drug (without the carrier), therefore allowingadministration of a higher dose of the drug in the conjugated form.

The term “therapeutic dose” as used herein means the dosage of a drug(without the carrier) that is acceptable for use clinically with respectto its toxicity.

In accordance with the present invention, the carrier may increase forexample, the potency of an anticancer drug, for example, an anticancerdrug which may be P-gp substrates.

In yet an additional aspect, the present invention more particularlyrelates to the use of a carrier, conjugate or the pharmaceuticalcomposition described herein for increasing (optimizing) an anti-tumorgrowth effect of an anticancer drug.

The present invention further provides the use of a carrier selectedfrom the group consisting of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2, biologically active analogs,derivatives or fragments and combination thereof for transporting a drugor a label at or to a desired target site or for transporting a drug ora label inside a target cell and/or for promoting the accumulation of adrug or a label inside a cell, such as for example, a cell whichexpresses P-gp at its surface or a cell which is able to express P-gp(at its surface).

The carrier may be used, for example, for promoting the accumulation,inside a cell, of a drug which may comprise the characteristic of beingexpulsed (i.e., expelled, transported outside a cell, ejected) by P-gpor a P-gp related protein.

In accordance with the present invention, the desired site may be, forexample and without limitation, the brain or other sites outside thebrain (e.g., an extracranial site) such as for example, the kidney, theliver, the pancreas, the colon, the eyes, the lungs and combinationthereof. Therefore, the desired target site may be one or more siteselected from the group consisting of the brain, the kidney, the liver,the pancreas, the colon, the eyes, the lungs and combination thereof.

In accordance with a particular embodiment of the present invention, adesired target site may be, for example, a brain cell or tissue.

In accordance with another particular embodiment of the presentinvention, a desired target site may be, for example, a liver cell ortissue.

In accordance with a further particular embodiment of the presentinvention, a desired target site may be for example, a kidney cell ortissue.

In accordance with yet a further particular embodiment of the presentinvention, a desired target site may be for example, a pancreas cell ortissue.

In accordance with another particular embodiment of the presentinvention, a desired target site may be for example, a colon cell ortissue.

In accordance with yet another particular embodiment of the presentinvention, a desired target site may be for example, eye or an eye cell.

In accordance with a further particular embodiment of the presentinvention, a desired target site may be for example, a lung cell ortissue.

Further in accordance with the present invention, the desired site maybe a site which comprises a cell expressing a carrier receptor ortransporter, for example, a cell expressing a low-density lipoproteinrelated receptor (LRP). The cell may also be a cell which co-expressesP-gp or a P-gp related protein. The cell may be, for example a normalcell, a tumor cell, or a metastatic cell. The carrier of the presentinvention may thus be used to target a brain cell, a liver cell, akidney cell, a pancreas cell, a colon cell, an eye cell, a lung cell andcombination thereof (either normal or tumoral).

The present invention also relates in a more particular aspect thereofto the use of a carrier described herein (e.g., which may be selectedfrom the group consisting of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2 and biologically active analogs,derivatives or fragments thereof) or the pharmaceutical composition orthe conjugate described herein for promoting intracellular accumulationof a compound (i.e., promoting accumulation of the compound inside acell).

In accordance with an embodiment of the present invention, the compoundmay be selected, for example, from the group consisting of a label, aprotein, a peptide and a small molecule drug.

In accordance with a further embodiment of the present invention, thecell may be a cell which is able to express P-gp or which expressesP-gp. More particularly the cell may express P-gp (MDR1) at the cellsurface.

The cell may be, for example, a tumor cell. The tumor cell may originatefor example and without limitation, from a brain tumor, a lung tumor, abreast tumor, a kidney tumor, an eye tumor, a liver tumor, a colorectaltumor, a tumor of an urinary organ, etc.

In accordance with the present invention the cell may be located outsideof a brain of an individual (mammal, animal, etc.). For example, thecell may be a tumor cell which may be located outside of a brain of anindividual (mammal, animal, etc.).

In accordance with a further embodiment of the present invention, thecell may be located inside a brain of an individual. The cell may be,for example, a tumor cell which may be located inside of a brain of anindividual (mammal, animal, etc.).

In an exemplary embodiment of the present invention, the tumor cell maybe a brain tumor cell. For example, the brain tumor cell may originatefrom a glioblastoma or may be a glioblastoma.

In another exemplary embodiment of the present invention, the tumor cellmay be a lung tumor cell.

The present invention also relates in an additional aspect thereof tothe use of the carrier, the conjugate or the pharmaceutical compositiondescribed herein for reducing the elimination of a drug from the insideof a cell, such as for example a cell which may be able to express P-gp(MDR1) or which expresses P-gp. In accordance with the presentinvention, the drug may be a P-gp substrate.

Also in accordance with the present invention, the cell may be amultiple drug resistant cancer cell.

In yet an additional aspect thereof, the present invention relates tothe use of a carrier, the conjugate or the pharmaceutical compositiondescribed herein for reducing the growth of a cell. For that purpose,the carrier may be conjugated with a drug which may be able to reducethe growth of a cell.

In accordance with a non-limitative exemplary embodiment of theinvention, the carrier, the conjugate thus formed or the pharmaceuticalcomposition may be used to reduce the growth of a tumor cell or anendothelial cell.

In a particular embodiment of the invention, the tumor cell may be ableto express or expresses P-gp (MDR1).

In an exemplary embodiment of the invention, the tumor cell may be abrain tumor cell. More specifically, the brain tumor cell may originatefrom a glioblastoma or may be a glioblastoma.

In another exemplary embodiment of the invention, the tumor cell may bea lung tumor cell.

In yet another exemplary embodiment of the invention, the tumor cell maybe a breast tumor cell.

In a further exemplary embodiment of the invention, the tumor cell maybe a kidney tumor cell.

In yet a further exemplary embodiment of the invention, the tumor cellmay be an eye tumor cell.

In an additional embodiment of the invention, the tumor cell may be froma colorectal cancer.

In another embodiment of the invention, the tumor cell may be from theliver.

In yet another additional embodiment of the invention, the tumor cellmay be from a urinary organ tumor.

In a particular embodiment of the invention, the anticancer drug may,more specifically be Taxol, Taxotere or a Taxol or Taxotere derivative.

In accordance with another embodiment of the present invention, theanticancer drug may be, for example, vinblastine.

In accordance with yet another embodiment of the present invention, theanticancer drug may be, for example, vincristine.

In accordance with a further embodiment of the present invention, theanticancer drug may be, for example, etoposide.

In accordance with a further embodiment of the present invention, theanticancer drug may be, for example, doxorubicin.

In accordance with an additional embodiment of the present invention,the anticancer drug may be, for example, cyclophosphamide.

In accordance with yet an additional embodiment of the presentinvention, the anticancer drug may be, for example, melphalan.

In accordance with yet another embodiment of the present invention, theanticancer drug may be, for example chlorambucil.

In another aspect, the present invention relates to the use of a carrierdescribed herein in the making of a pharmaceutical composition ormedicament for modifying the pharmacokinetics of the small moleculedrug.

More particularly the carrier may be used for reducing the growth of acell.

Also more particularly, the carrier may be used for promotingaccumulation of the small molecule drug inside a cell.

In addition the carrier may be used for reducing the elimination of thesmall molecule drug from the inside of a cell.

Also, the carrier may be used for increasing an anti-tumor growth effectof the small molecule drug.

Furthermore, the carrier may be used to improve the bioavailability ofthe small molecule drug.

In addition and in accordance with the present invention, the carriermay be used to change the (usual) tissue distribution of the smallmolecule drug.

In addition, the present invention relates to the use of a carrierselected from the group consisting of aprotinin, a biologically activeaprotinin fragment, Angiopep-1, Angiopep-2, biologically active analogand combination thereof for treating cancer, metastatic cancer and/ormetastasis. In accordance with the present invention, an exemplarymetastasis may comprise, without limitation, a metastasis which mayoriginate from a breast tumor, a lung tumor, a melanoma, etc.

The present invention also relates to the use of a carrier selected fromthe group consisting of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2, biologically active analogs,derivatives or fragments and combination thereof for the detection of adesired target cell.

The present invention further provides the use of a carrier selectedfrom the group consisting of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2, biologically active analogs,derivatives or fragments and combination thereof for the diagnostic of acancer, a metastatic cancer and/or metastasis.

The present invention additionally relates in a further aspect, to acomposition (e.g., pharmaceutical composition) comprising a carrier(and/or pharmaceutically acceptable salt thereof) of the presentinvention and a pharmaceutically acceptable carrier.

The present invention in an additional aspect relates to a conjugateand/or pharmaceutically acceptable salt thereof. The conjugate maycomprise, for example, a carrier as described herein and a drug, a labelor a protein. The carrier may be covalently attached to the drug, label,protein or peptide.

More particularly, the conjugate may comprise, for example a carrierselected from the group consisting of aprotinin, a biologically activeaprotinin fragment, Angiopep-1, Angiopep-2, biologically active analogs,derivatives or fragments and combination thereof and a compound selectedfrom the group consisting of a drug, a label, a protein and combinationthereof. In accordance with the present invention, the conjugate maycomprise one or more drug molecules.

Also in accordance with the present invention, the compound may or maynot be released from the carrier. The compound may therefore bereleasable from the conjugate (or from the carrier).

In accordance with the present invention, the conjugate may comprise theformula R-L-M wherein R is a class of molecules related to aprotinin(e.g., aprotinin, aprotinin fragment, Angiopep-1, Angiopep-2, analogs,derivatives or fragments), L may be a linker or a bond and M may be anagent or a drug selected from the group consisting of a drug (e.g., asmall molecule drug), a label, a protein (e.g., antibody, an antibodyfragment) and a polypeptide. It is to be understood herein that theformula R-L-M is not intended to be restricted to a specific order orspecific ratio. As being exemplified herein, M may be found in severalratios over R.

The present invention relates in a further aspect thereof to the use ofa conjugate which may comprise a) a carrier which may be selected fromthe group consisting of aprotinin, a biologically active aprotininfragment, Angiopep-1, Angiopep-2 and biologically active analogs,derivatives or fragments thereof and b) at least one small molecule drugor label for modifying the pharmacokinetics (in vivo) of the drug orlabel which is attached thereto.

In accordance with one embodiment of the present invention, theconjugate may be, more particularly, used for reducing the growth of acell.

Further in accordance with the present invention, the conjugate may beused for promoting accumulation of the small molecule drug or labelinside a cell.

Also in accordance with the present invention, the conjugate may also beused for reducing the elimination of the small molecule drug or labelfrom the inside of a cell.

Further in accordance with the present invention, the conjugate may beused for increasing an anti-tumor growth effect of the small moleculedrug.

Also in accordance with the present invention, the conjugate may improvethe bioavailability of the compound.

Further in accordance with the present invention, the conjugate may alsochange the tissue distribution of the compound.

In one embodiment of the invention, the small molecule drug may be ableto reduce the growth of a cell.

The present invention also provides in an additional aspect thereof forthe use of a conjugate which may comprise a) a carrier which may beselected, for example, from the group consisting of aprotinin, abiologically active aprotinin fragment, Angiopep-1, Angiopep-2 andbiologically active analogs, derivatives or fragments thereof and b) atleast one small molecule drug in the making (manufacture) of apharmaceutical composition or medicament for modifying thepharmacokinetics of the small molecule drug.

The present invention also relates to the use of the carrier in themanufacture of a composition or medicament for the treatment of acondition such as cancer, metastatic cancer and/or metastasis.

The present invention also provides in an additional aspect thereof forthe use of a conjugate which may comprise a) a carrier which may beselected, for example, from the group consisting of aprotinin, abiologically active aprotinin fragment, Angiopep-1, Angiopep-2 andbiologically active analogs, derivatives or fragments thereof and b) atleast one label, for the detection of a cell (e.g., tumor cell) whichmay be selected, for example, from the group consisting of an eye cell,a brain cell, a breast cell, a liver cell a kidney cell, a urinary organcell, a colon cell, a cell from the rectum and a lung cell.

In a particular embodiment of the present invention, the cell which maybe detected may be, for example, an eye cell.

In another particular embodiment of the present invention, the cellwhich may be detected may be, for example, a brain cell.

In an additional particular embodiment of the present invention, thecell which may be detected may be, for example, a liver cell.

In yet an additional particular embodiment of the present invention, thecell which may be detected may be, for example, a breast cell.

In another particular embodiment of the present invention, the cellwhich may be detected may be, for example, a kidney cell.

In yet another particular embodiment of the present invention, the cellwhich may be detected may be, for example, lung cell. The presentinvention additionally relates to a composition (e.g., pharmaceuticalcomposition) comprising a conjugate of the present invention and apharmaceutically acceptable carrier.

The pharmaceutical composition described herein may be used, forexample, in the treatment or detection of cancer, a metastatic cancerand/or metastasis.

The present invention provides in a particular aspect thereof, apharmaceutical composition which may be able, for example, of reducingthe growth of a cell (e.g., tumor cell) or for the detection of a cell(e.g., tumor cell), the pharmaceutical composition may comprise:

-   -   a) a conjugate which may comprise a carrier selected from the        group consisting of aprotinin, a biologically active aprotinin        fragment, Angiopep-1, Angiopep-2 and biologically active        analogs, derivatives or fragments thereof and a label or a small        molecule drug able to reduce the growth of a cell (e.g., tumor        cell) and;    -   b) a pharmaceutically acceptable carrier.

The present invention also provides in a further aspect thereof, apharmaceutical composition which may comprise:

-   -   a) a conjugate which may comprise a carrier selected from the        group consisting of aprotinin, a biologically active aprotinin        fragment, Angiopep-1, Angiopep-2 and biologically active        analogs, derivatives or fragments thereof and a small molecule        drug or a label,    -   b) a pharmaceutically acceptable carrier, and;    -   c) a solubilizer.

In accordance with the present invention, the solubilizer may be, forexample, a poly-oxyethylene ester of fatty acid. Solubilizersencompassed by the present invention, include, for example, Solutol®HS-15.

An exemplary embodiment of the present invention, a suitable solubilizermay comprise, without limitation, a poly-oxyethylene ester of fatty acidsuch as for example, Solutol® HS-15.

In accordance with an embodiment of the invention, the pharmaceuticalcomposition may be used more specifically for modifying thepharmacokinetics of a compound, for reducing the growth of a tumor cellor for the detection of a tumor cell, etc.

The present invention further relates to the use of at least oneconjugate of the present invention for treating cancer, metastaticcancer and/or metastasis. In accordance with the present invention, anexemplary metastasis which may be treated using the conjugate of thepresent invention is a metastasis which may originate, for example andwithout limitation from a breast tumor, a lung tumor, a melanoma etc.

The present invention also provides in another aspect thereof, a methodfor treating a patient having a cancer (such as, for example, a primarytumor or a metastatic cancer and/or a metastasis) or for detecting acancer cell, the method may comprise administering an individual with apharmaceutical composition described herein or with a conjugate whichmay comprise a) a carrier which may be selected from the groupconsisting of aprotinin, a biologically active aprotinin fragment,Angiopep-1, Angiopep-2 and biologically active analogs, derivatives orfragments thereof and b) an anticancer drug or a label.

In accordance with the present invention, the individual may have, forexample, an extracranial tumor, a primary brain tumor or a brain tumorof metastatic origin.

The method may also comprise a step of assessing whether the tumor ofthe individual comprises a multiple drug resistant tumor cell or forexample a cell expressing P-gp (MDR1) or determining whether the tumormay have or has a multiple resistance drug phenotype.

The individual may be one which has a tumor. The individual may also beone which has received or which will receive chemotherapy orradiotherapy or both. The individual may also be one who has hadsurgery. Furthermore, the individual may be one that has a brain tumoror may also be one who has a tumor at another site than brain (is freeof a brain tumor). An individual in need may also be, for example, anindividual which present or is at risk of presenting, a resistance (amultiple drug resistance (MDR) phenotype) to at least one drug.

More particularly and in accordance with the present invention, theindividual may have, for example, an extracranial tumor.

In accordance with the present invention, the extracranial tumor may be,for example, a lung tumor.

Also in accordance with the present invention, the extracranial tumormay be, for example, an extracranial metastasis from a brain tumor. In amore specific embodiment of the present invention, the extracranialbrain tumor may be, for example, a glioblastoma (extracranial metastasisfrom glioblastoma).

In another particular embodiment of the present invention, theindividual may have a brain tumor of metastatic origin. The brain tumorof metastatic origin may originate, for example, from a lung tumor. Thebrain tumor of metastatic origin may also originate, for example, from abreast tumor. Additionally, the brain tumor of metastatic origin mayalso originate, for example, from a melanoma. Furthermore and asdescribed herein, the brain tumor of metastatic origin may alsooriginate, for example, from a colorectal cancer. In addition, the braintumor of metastatic origin may also originate, for example, from aurinary organ tumor.

In accordance with the present invention, the tumor may comprise a tumorcell which may be able to express P-gp or which expresses P-gp.

In accordance with the present invention the tumor may comprise a tumorcell which may be able to express LRP or which expresses LRP.

In accordance with the present invention, the tumor cell may comprise atumor cell which may be able to co-express P-gp and LRP or whichco-expresses P-gp and LRP. P-gp and LRP may be located, for example, ata cell surface.

More particularly, the present invention in an aspect thereof, relatesto a method of promoting accumulation of a drug in the brain of anindividual having a metastasis. The method may comprise administering acarrier or conjugate as described herein to an individual having a brainmetastasis. In accordance with the present invention, the metastasis mayoriginate from a lung cancer, etc.

The present invention provides in an additional aspect thereof, a methodfor promoting intracellular accumulation of a compound selected from thegroup consisting of a label, a protein, a peptide and a small moleculedrug.

The method may comprise the step of contacting the cell or providing thecell with a conjugate which may comprise a) a carrier which may beselected from the group consisting of aprotinin, a biologically activeaprotinin fragment, Angiopep-1, Angiopep-2 and biologically activeanalogs, derivatives or fragments thereof and b) the desired compound.

In accordance with the present invention, the cell may be, for example,a cell expressing P-gp.

Further in accordance with the present invention, the cell may be, forexample, a brain cell, a lung cell, a breast cell, a kidney cell, an eyecell or a liver cell.

Also in accordance with the present invention, the cell may be, forexample, a tumor cell which may be located outside of a brain of anindividual (mammal, animal, etc.).

The present invention also provides in yet an additional aspect, amethod for reducing the elimination of a drug from the inside of a cellwhich is able to express or which expresses P-gp (MDR1).

The method may comprise conjugating the drug with a carrier describedherein, thereby forming a conjugate and providing the cell with theconjugate.

In an exemplary embodiment of the invention, the cell may be a multipledrug resistant cancer cell.

In a further exemplary embodiment of the invention, the cell may becomprised within an extracranial tumor of an individual.

In an additional exemplary embodiment of the invention, the cell may becomprised within a brain of an individual. The cell may be a tumor cell,such as for example a brain tumor cell (primary) or a metastatic braintumor cell.

Means for providing a cell with a conjugate is, for example, to providethe conjugate to an individual who comprises a multiple drug resistantcell or a multiple drug resistant tumor cell (e.g. or a cell whichexpresses MDR1).

In accordance with the present invention the method may be used toreduce elimination of a drug which is a P-gp substrate or which may be aP-gp substrate.

In another aspect, the present invention provides a method for reducingthe growth of a cell. The method may comprise contacting the cell with aconjugate which may comprise a) a carrier which may be selected from thegroup consisting of aprotinin, a biologically active aprotinin fragment,Angiopep-1, Angiopep-2 and biologically active analogs, derivatives and;b) a drug able to reduce the growth of a cell, or with thepharmaceutical composition described herein.

In accordance with the present invention the carrier may be used toincrease the potency (efficiency, effectiveness) of the small moleculedrug.

Also in accordance with the present invention, conjugation of the smallmolecule drug with the carrier may be achieved through several meanswhich may include using a linker (e.g., a linker able to generate anester bond) which may allow association of the drug through a suitableatom, for example, through an oxygen atom.

In an additional aspect the present invention provides a method formodifying the pharmacokinetics of a compound which may be selected, forexample, from the group consisting of a label, a protein, a peptide anda small molecule drug, the method may comprise the step of conjugatingthe compound with a carrier described herein thereby forming a conjugateand providing the conjugate to an individual in need.

In accordance with the present invention, a ratio of one molecule of thecompound for each carrier molecule may be used for the conjugation step.

Further in accordance with the present invention, a ratio of at leasttwo molecules of the compound for each carrier molecule may be used forthe conjugation step.

Also in accordance with the present invention, a ratio of at least threemolecules of the compound for each carrier molecule may be used for theconjugation step.

In a particular embodiment of the invention, the compound may be, forexample, a small molecule drug.

In another particular embodiment of the invention, the compound may be,for example, a label.

In accordance with the present invention, the individual in need may be,more particularly, an individual having a tumor. For example, theindividual may have a brain tumor. In accordance with the presentinvention, the brain tumor may be, for example, a primary brain tumor.Also in accordance with the present invention, the brain tumor may be,for example, of a different tissue origin than brain tissue.

In an exemplary embodiment of the invention, the brain tumor of theindividual may be, for example, an extracranial brain tumor.

In accordance with the present invention, the tumor may comprise a tumorcell expressing or which is able to express P-gp (MDR1).

The brain tumor may originate, for example, from a lung tumor. The braintumor may also originate, for example, from a breast tumor. Also, forexample, the brain tumor may originate from a melanoma.

In accordance with a particular embodiment of the present invention, themethod may increase (optimize), for example, an anti-tumor growth effectof an anticancer drug (as compared with unconjugated drug).

In accordance with another particular embodiment of the presentinvention, the method may promote, for example, the accumulation of thecompound within a cell (i.e., inside a cell).

In accordance with yet another particular embodiment of the presentinvention, the method may allow a reduction in the elimination of thesmall molecule drug from the inside of a cell (e.g., a cell expressingP-gp).

In accordance with an additional embodiment of the present invention,the method may allow a reduction of cell growth (e.g., reduction oftumor cell growth).

Furthermore, the method may allow an improvement in the bioavailabilityof the small molecule drug.

In addition and in accordance with the present invention, the method maybe used to change the (usual) tissue distribution of the small moleculedrug.

In yet a further aspect, the present invention provides for the use of acarrier, conjugate or the pharmaceutical composition as described hereinfor reducing LRP-dependent accumulation of RAP and for reducingRAP-mediated cellular (e.g., intracellular) event or effect.

For the purpose of the present invention the following terms are definedbelow.

The term “Angiopep” as used herein refers to Angiopep-1 and Angiopep-2.

The terms “Taxol-Angiopep” and “TxlAn” are used interchangeably andrefer to Taxol-Angiopep-1 and Taxol-Angiopep-2, comprising 1, 2 or 3Taxol molecules.

The terms “Taxol-Angiopep-1” and “TxlAn1” are used interchangeably andrefer to Taxol-Angiopep-1, comprising either 1, 2 or 3 Taxol molecules.

The terms “Taxol-Angiopep-2” and “TxlAn2” are used interchangeably andrefer to Taxol-Angiopep-2, comprising either 1, 2 or 3 Taxol molecules.

The term “TxlAn1 (2:1)” refers to the ratio of Taxol over Angiopep-1molecules in a given conjugate. For example, the term “TxlAn1 (2:1)relates to a conjugate having 2 molecules of Taxol associated with onemolecule of Angiopep-1. In addition, the term “TxlAn1 (3:1) relates to aconjugate having 3 molecules of Taxol associated with one molecule ofAngiopep-1.

Similarly, the term “TxlAn2 (2:1)” refers to the ratio of Taxol overAngiopep-2 molecules in a given conjugate. For example, the term “TxlAn2(2:1) relates to a conjugate having 2 molecules of Taxol associated withone molecule of Angiopep-2. In addition, the term “TxlAn2 (3:1) relatesto a conjugate having 3 molecules of Taxol associated with one moleculeof Angiopep-2.

The term “carrier” or “vector” is intended to mean a compound ormolecule that is able to transport a molecule at a desired targeted siteor cell. A carrier may be attached to (covalently or not) or conjugatedto another compound or agent and thereby may be able to transport theother compound or agent a desired targeted site. The carrier may be, butis not limited to, a protein, a peptide or to a peptidomimetic and canbe naturally occurring or produced by chemical synthesis or recombinantgenetic technology (genetic engineering).

The expression “small molecule drug” is intended to mean a drug having amolecular weight of 1000 g/mol or less or between 300 and 700 g/mol.

The terms “treatment”, “treating” and the like are intended to meanobtaining a desired pharmacologic and/or physiologic effect, e.g.,inhibition of cancer cell growth, death of a cancer cell or ameliorationof a neurological disease or condition. The effect may be prophylacticin terms of completely or partially preventing a disease or symptomthereof and/or may be therapeutic in terms of a partial or complete curefor a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing a disease(e.g., preventing cancer) or condition from occurring in an individualwho may be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting a disease, (e.g., arresting its development);or (c) relieving a disease (e.g., reducing symptoms associated with adisease). “Treatment” as used herein covers any administration of apharmaceutical agent or compound to an individual to treat, cure,alleviate, improve, diminish or inhibit a condition in the individual,including, without limitation, administering a drug comprising a carrierdescribed herein or a conjugate to an individual in need thereof.

The term “cancer” is intended to mean any cellular malignancy whoseunique trait is the loss of normal controls which results in unregulatedgrowth, lack of differentiation and ability to invade local tissues andmetastasize. Cancer can develop in any tissue of any organ. Morespecifically, cancer is intended to include, without limitation, cancerof the brain.

The term “administering” and “administration” is intended to mean a modeof delivery including, without limitation, intra-arterially,intra-nasally, intra-peritoneally, intravenously, intramuscularly,sub-cutaneously, transdermally or per os. A daily dosage may be dividedinto one, two or more doses in a suitable form to be administered atone, two or more times throughout a time period.

The term “therapeutically effective” or “effective amount” is intendedto mean an amount of a compound sufficient to substantially improve somesymptom associated with a disease or a medical condition. For example,in the treatment of cancer, an agent or compound which decreases,prevents, delays, suppresses, or arrests any symptom of the disease orcondition would be therapeutically effective. A therapeuticallyeffective amount of an agent or compound is not required to cure adisease or condition but will provide a treatment for a disease orcondition such that the onset of the disease or condition is delayed,hindered, or prevented, or the disease or condition symptoms areameliorated, or the term of the disease or condition is changed or, forexample, is less severe or recovery is accelerated in an individual.

The carrier and conjugate of the present invention may be used incombination with either conventional methods of treatment and/or therapyor may be used separately from conventional methods of treatment and/ortherapy.

When the conjugates of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a carrier-agent conjugate of the present invention inassociation with a pharmaceutically acceptable carrier orpharmaceutically acceptable excipient, as described herein, and anothertherapeutic or prophylactic agent known in the art.

Pharmaceutically acceptable acid (addition) salts may be prepared bymethods known and used in the art.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the agent together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvant and/orcarriers. A “therapeutically effective amount” as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen. Such compositions are liquids or lyophilizedor otherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts).Solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines). Other embodiments of thecompositions of the invention incorporate particulate forms protectivecoatings, protease inhibitors or permeation enhancers for various routesof administration, including parenteral, pulmonary, nasal, oral,vaginal, rectal routes. In one embodiment the pharmaceutical compositionis administered parenterally, paracancerally, transmucosally,transdermally, intramuscularly, intravenously, intradermally,subcutaneously, intraperitonealy, intraventricularly, intracranially andintratumorally.

Further, as used herein “pharmaceutically acceptable carrier” or“pharmaceutical carrier” are known in the art and include, but are notlimited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline.Additionally, such pharmaceutically acceptable carriers may be a buffer,an aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

A “fragment” is to be understood herein as a polypeptide originatingfrom (encompassing) a portion of an original or parent sequence.Fragments encompass polypeptides having truncations of one or more aminoacids, wherein the truncation may originate from the amino terminus(N-terminus), carboxy terminus (C-terminus), or from the interior of theprotein. A fragment may comprise the same sequence as the correspondingportion of the original sequence. Biologically active fragments of thecarrier described herein are encompassed by the present invention.

A “derivative” in the context of proteins or peptides is to beunderstood herein as a polypeptide originating from an original sequenceor from a portion of an original sequence and which may comprise one ormore modification; for example, one or more modification in the aminoacid sequence (e.g., an amino acid addition, deletion, insertion,substitution etc.) and one or more modification in the backbone orside-chain of one or more amino acid, or an addition of a group oranother molecule to one or more amino acids (side-chains or backbone).Biologically active derivatives of the carrier described herein areencompassed by the present invention.

An “analogue” in the context of proteins or peptides is to be understoodherein as a molecule having a biological activity and chemical structuresimilar to that of a polypeptide described herein. An analog comprises apolypeptide which may have, for example, one or more amino acidinsertion, either at one or both of the end of the polypeptide and/orinside the amino acid sequence of the polypeptide.

An “analogue” may have sequence similarity with that of an originalsequence or a portion of an original sequence and may also have amodification of its structure as discussed herein. For example, an“analogue” may have at least 90% sequence similarity with an originalsequence or a portion of an original sequence. An “analogue” may alsohave, for example; at least 70% or even 50% sequence similarity with anoriginal sequence or a portion of an original sequence. An “analogue”may have, for example, 50%, 70%, 80% or 90% sequence similarity to anoriginal sequence with a combination of one or more modification in abackbone or side-chain of an amino acid, or an addition of a group oranother molecule, etc. Amino acids which are intended to be similar (aconservative amino acid) to another are known in the art and includes,for example, those listed in Table 1.

In addition, an “analogue” may have at least 50%, 70%, 80% or 90%sequence identity with an original sequence or a portion of an originalsequence. Also, an “analogue” may have, for example, 50%, 70%, 80% or90% sequence identity to an original sequence with a combination of oneor more modification in a backbone or side-chain of an amino acid, or anaddition of a group or another molecule, etc.

Similarity or identity may be compared, for example, over a region of 2,3, 4, 5, 10, 19, 20 amino acids or more (and any number therebetween).Identity may include herein, amino acids which are identical to theoriginal peptide and which may occupy the same or similar position whencompared to the original polypeptide. An analog which have, for example,50% identity with an original polypeptide may include for example, ananalog comprising 50% of the amino acid of the original polypeptide andsimilarly with the other percentages. It is to be understood herein thatgaps may be found between the amino acids of an analogs which areidentical or similar to amino acids of the original peptide. The gapsmay include no amino acids, one or more amino acids which are notidentical or similar to the original peptide. Biologically activeanalogs of the carrier of the present invention are encompassedherewith.

Thus, biologically active polypeptides in the form of the originalpolypeptides, fragments (modified or not), analogues (modified or not),derivatives (modified or not), homologues, (modified or not) of thecarrier described herein are encompassed by the present invention.

Therefore, any polypeptide having a modification compared to an originalpolypeptide which does not destroy significantly a desired biologicalactivity is encompassed herein. It is well known in the art, that anumber of modifications may be made to the polypeptides of the presentinvention without deleteriously affecting their biological activity.These modifications may, on the other hand, keep or increase thebiological activity of the original polypeptide or may optimize one ormore of the particularity (e.g. stability, bioavailability, etc.) of thepolypeptides of the present invention which, in some instance might beneeded or desirable. Polypeptides of the present invention comprises forexample, those containing amino acid sequences modified either bynatural processes, such as posttranslational processing, or by chemicalmodification techniques which are known in the art. Modifications mayoccur anywhere in a polypeptide including the polypeptide backbone, theamino acid side-chains and the amino- or carboxy-terminus. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications. Polypeptidesmay be branched as a result of ubiquitination, and they may be cyclic,with or without branching. Cyclic, branched and branched cyclicpolypeptides may result from posttranslational natural processes or maybe made by synthetic methods. Modifications comprise for example,without limitation, pegylation, acetylation, acylation, addition ofacetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation,biotinylation, carbamoylation, carboxyethylation, esterification,covalent attachment to flavin, covalent attachment to a heme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of drug, covalent attachment of a marker (e.g., fluorescent,radioactive, etc.), covalent attachment of a lipid or lipid derivative,covalent attachment of phosphatidylinositol, cross-linking, cyclization,disulfide bond formation, demethylation, formation of covalentcross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation and ubiquitination, etc. It is to beunderstood herein that more than one modification to the polypeptidesdescribed herein are encompassed by the present invention to the extentthat the biological activity is similar to the original (parent)polypeptide.

As discussed above, polypeptide modification may comprise, for example,amino acid insertion (i.e., addition), deletion and substitution (i.e.,replacement), either conservative or non-conservative (e.g., D-aminoacids, desamino acids) in the polypeptide sequence where such changes donot substantially alter the overall biological activity of thepolypeptide.

Example of substitutions may be those, which are conservative (i.e.,wherein a residue is replaced by another of the same general type orgroup) or when wanted, non-conservative (i.e., wherein a residue isreplaced by an amino acid of another type). In addition, a non-naturallyoccurring amino acid may substitute for a naturally occurring amino acid(i.e., non-naturally occurring conservative amino acid substitution or anon-naturally occurring non-conservative amino acid substitution).

As is understood, naturally occurring amino acids may be sub-classifiedas acidic, basic, neutral and polar, or neutral and non-polar.Furthermore, three of the encoded amino acids are aromatic. It may be ofuse that encoded polypeptides differing from the determined polypeptideof the present invention contain substituted codons for amino acids,which are from the same type or group as that of the amino acid to bereplaced. Thus, in some cases, the basic amino acids Lys, Arg and Hismay be interchangeable; the acidic amino acids Asp and Glu may beinterchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, andAsn may be interchangeable; the non-polar aliphatic amino acids Gly,Ala, Val, Ile, and Leu are interchangeable but because of size Gly andAla are more closely related and Val, Ile and Leu are more closelyrelated to each other, and the aromatic amino acids Phe, Trp and Tyr maybe interchangeable.

It should be further noted that if the polypeptides are madesynthetically, substitutions by amino acids, which are not naturallyencoded by DNA (non-naturally occurring or unnatural amino acid) mayalso be made.

A non-naturally occurring amino acid is to be understood herein as anamino acid which is not naturally produced or found in a mammal. Anon-naturally occurring amino acid comprises a D-amino acid, an aminoacid having an acetylaminomethyl group attached to a sulfur atom of acysteine, a pegylated amino acid, etc. The inclusion of a non-naturallyoccurring amino acid in a defined polypeptide sequence will thereforegenerate a derivative of the original polypeptide. Non-naturallyoccurring amino acids (residues) include also the omega amino acids ofthe formula NH₂(CH₂)_(n)COOH wherein n is 2-6, neutral nonpolar aminoacids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methylisoleucine, norleucine, etc. Phenylglycine may substitute for Trp, Tyror Phe; citrulline and methionine sulfoxide are neutral nonpolar,cysteic acid is acidic, and omithine is basic. Proline may besubstituted with hydroxyproline and retain the conformation conferringproperties.

It is known in the art that analogues may be generated by substitutionalmutagenesis and retain the biological activity of the polypeptides ofthe present invention. These analogues have at least one amino acidresidue in the protein molecule removed and a different residue insertedin its place. Examples of substitutions identified as “conservativesubstitutions” are shown in Table 1. If such substitutions result in achange not desired, then other type of substitutions, denominated“exemplary substitutions” in Table 1, or as further described herein inreference to amino acid classes, are introduced and the productsscreened.

In some cases it may be of interest to modify the biological activity ofa polypeptide by amino acid substitution, insertion, or deletion. Forexample, modification of a polypeptide may result in an increase in thepolypeptide's biological activity, may modulate its toxicity, may resultin changes in bioavailability or in stability, or may modulate itsimmunological activity or immunological identity. Substantialmodifications in function or immunological identity are accomplished byselecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation. (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. Naturally occurring residues are dividedinto groups based on common side chain properties:

-   -   (1) hydrophobic: norleucine, methionine (Met), Alanine (Ala),        Valine (Val), Leucine (Leu), Isoleucine (Ile)    -   (2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine        (Thr)    -   (3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)    -   (4) basic: Asparagine (Asn), Glutamine (Gln), Histidine (His),        Lysine (Lys), Arginine (Arg)    -   (5) residues that influence chain orientation: Glycine (Gly),        Proline (Pro); and aromatic: Tryptophan (Trp), Tyrosine (Tyr),        Phenylalanine (Phe)

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another.

TABLE 1 amino acid substitution Original residue Exemplary substitutionConservative substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gln, Lys,Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu norleucine Leu (L)Norleucine, Ile, Val, Met, Ile Ala, Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser(S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, SerPhe Val (V) Ile, Leu, Met, Phe, Ala, Leu norleucine

A biologically active analog may be, for example, an analogue having atleast one conservative amino acid substitution in the original sequence.A biologically active analog may also be for example, an analog havingan insertion of an amino acid.

For example, an Angiopep-1 analog may have the formula I:X₁-Angiopep-1-X₂

For example, an Angiopep-2 analog may have the formula II:X₁-Angiopep-2-X₂

X₁ and X₂ may independently be an amino acid sequence of from between 0to about 100 (e.g., from between 0 to about 60) amino acids. X₁ and X₂may be derived from consecutive amino acids of aprotinin or aprotininanalogs (homologous amino acid sequence) or may be any other amino acidsequence (heterologous amino acid sequence).

A compound of either formula I or II, may also comprises an amino acidsubstitution, deletion or insertion within the amino acid sequence ofAngiopep-1 or Angiopep-2. The analog however would preferably bebiologically active as determined by one of the assays described hereinor by any similar or equivalent assays.

Examples of aprotinin analogs may be found by performing a protein blast(Genebank: www.ncbi.nlm.nih.gov/BLAST/) of the synthetic aprotininsequence (or portion thereof) disclosed in international application no.PCT/CA2004/000011. Exemplary aprotinin analogs may be found, for exampleunder accession nos. CAA37967 (GI:58005), 1405218C (GI:3604747) etc.

A biologically active fragment of a polypeptide (e.g., of 19 aminoacids) described herein may include for example a polypeptide of fromabout 7, 8, 9 or 10 to 18 amino acids.

A biologically active polypeptide (e.g., carrier) may be identified byusing one of the assays or methods described herein. For example acandidate carrier may be produced by conventional peptide synthesis,conjugated with Taxol as described herein and tested in an in vivo modelas described in Example 5. A biologically active carrier may beidentified, for example, based on its efficacy at reducing tumor burdencompared to placebo-treated mice. A small molecule drug candidate whichmay be used in the conjugation of a carrier described herein may beidentified, for example, by determining or not whether the drug isexpulsed from P-gp over-expressing cells as described herein andevaluating whether its conjugation to the carrier increases itsaccumulation inside a desired cell.

Examples of biologically active carrier (i.e., biologically activeanalog of Angiopep-1 and/or Angiopep-2) may include for example,peptides derived from the kunitz-domain such as TFFYGGCRGKRNNFKTKEY (SEQID NO: 76), RFKYGGCLGNKNNYLRLKY (SEQ ID NO: 91) and TFFYGGCRAKRNNFKRAKY(SEQ ID NO: 5). Other examples of biologically active analogs may befound in Table 2A and 2B and in the Sequence Listing.

TABLE 2A Design of 96 peptides from similar domain to aprotinin andAngiopep-1 with different charges and amino acid insertions (SEQ IDNOS:1-96) 96 PEPTIDES ORDERED AT SYNPEP (California, USA) #Pep SEQ IDProteins Characteristics NO: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 Aprot-synth CHARGE (+6) 1 T F V Y G G C R A K R N N F K SA E D Blkunin HI-30 2 T F Q Y G G C M G N G N N F V T E K E Amyloid 3 PF F Y G G C G G N R N N F D T E E Y Kunitz-Inhib 1 4 S F Y Y G G C L G NK N N Y L R E E E Peptides 5 T F F Y G G C R A K R N N F K R A K Y 6 T FF Y G G C R G K R N N F K R A K Y 7 T F F Y G G C R A K K N N Y K R A KY 8 T F F Y G G C R G K K N N F K R A K Y 9 T F Q Y G G C R A K R N N FK R A K Y 10 T F Q Y G G C R G K K N N F K R A K Y CHARGE (+5) 11 T F FY G G C L G K R N N F K R A K Y 12 T F F Y G G S L G K R N N F K R A K Y13 P F F Y G G C G G K K N N F K R A K Y 14 T F F Y G G C R G K G N N YK R A K Y 15 P F F Y G G C R G K R N N F L R A K Y 16 T F F Y G G C R GK R N N F K R E K Y 17 P F F Y G G C R A K K N N F K R A K E 18 T F F YG G C R G K R N N F K R A K D CHARGE (+4) 19 T F F Y G G C R A K R N N FD R A K Y 20 T F F Y G G C R G K K N N F K R A E Y 21 P F F Y G G C G AN R N N F K R A K Y 22 T F F Y G G C G G K K N N F K T A K Y 23 T F F YG G C R G N R N N F L R A K Y 24 T F F Y G G C R G N R N N F K T A K Y25 T F F Y G G S R G N R N N F K T A K Y CHARGE (+3) 26 T F F Y G G C LG N G N N F K R A K Y 27 T F F Y G G C L G N R N N F L R A K Y 28 T F FY G G C L G N R N N F K T A K Y 29 T F F Y G G C R G N G N N F K S A K Y30 T F F Y G G C R G K K N N F D R E K Y 31 T F F Y G G C R G K R N N FL R E K E 32 T F F Y G G C R G K G N N F D R A K Y 33 T F F Y G G S R GK G N N F D R A K Y CHARGE (+2) 34 T F F Y G G C R G N G N N F V T A K Y35 P F F Y G G C G G K G N N Y V T A K Y 36 T F F Y G G C L G K G N N FL T A K Y 37 S F F Y G G C L G N K N N F L T A K Y HUMAN 38 T F F Y G GC G G N K N N F V R E K Y HUMAN 39 T F F Y G G C M G N K N N F V R E K YHUMAN 40 T F F Y G G S M G N K N N F V R E K Y HUMAN 41 P F F Y G G C LG N R N N Y V R E K Y HUMAN 42 T F F Y G G C L G N R N N F V R E K YHUMAN 43 T F F Y G G C L G N K N N Y V R E K Y CHARGE (+1) 44 T F F Y GG C G G N G N N F L T A K Y 45 T F F Y G G C R G N R N N F L T A E Y 46T F F Y G G C R G N G N N F K S A E Y 47 P F F Y G G C L G N K N N F K TA E Y 48 T F F Y G G C R G N R N N F K T E E Y 49 T F F Y G G C R G K RN N F K T E E D HUMAN 50 P F F Y G G C G G N G N N F V R E K Y HUMAN 51S F F Y G G C M G N G N N F V R E K Y HUMAN 52 P F F Y G G C G G N G N NF L R E K Y HUMAN 53 T F F Y G G C L G N G N N F V R E K Y HUMAN 54 S FF Y G G C L G N G N N Y L R E K Y HUMAN 55 T F F Y G G S L G N G N N F VR E K Y CHARGE (+0) 56 T F F Y G G C R G N G N N F V T A E Y 57 T F F YG G C L G K G N N F V S A E Y 58 T F F Y G G C L G N R N N F D R A E YHUMAN 59 T F F Y G G C L G N R N N F L R E E Y HUMAN 60 T F F Y G G C LG N K N N Y L R E E Y HUMAN 61 P F F Y G G C G G N R N N Y L R E E YHUMAN 62 P F F Y G G S G G N R N N Y L R E E Y Aprotinin vs APROTININ 63M R P D F C L E P P Y T G P C V A R I M-term (1 helix α, A-term) 64 A RI I R Y F Y N A K A G L C Q T F V Y G (2β sheets, Y-term) 65 Y G G C R AK R N N Y K S A E D C M R T C G (1α, 1β) 66 P D F C L E P P Y T G P C VA R I I R Y F Y AngloPep AngloPep-1 67 T F F Y G G C R G K R N N F K T EE Y AngloPEP1 (lysine) 68 K F F Y G G C R G K R N N F K T E E YAngloPEP1 (4Y) 69 T F Y Y G G C R G K R N N Y K T E E Y cys bridge 70 TF F Y G G S R G K R N N F K T E E Y cys-Nterminal 71 C T F F Y G C C R GK R N N F K T E E Y cys-Cterminal 72 T F F Y G G C R G K R N N F K T E EY C cys-Nterminal 73 C T F F Y G S C R G K R N N F K T E E Ycys-Cterminal 74 T F F Y G G S R G K R N N F K T E E Y C pro 75 P F F YG G C R G K R N N F K T E E Y charge (+3) 76 T F F Y G G C R G K R N N FK T K E Y charge (+3)-cys 77 T F F Y G G K R G K R N N F K T E E Ycharge (+4) 78 T F F Y G G C R G K R N N F K T K R Y charge (+4)-cys 79T F F Y G G K R G K R N N F K T A E Y charge (+5) 80 T F F Y G G K R G KR N N F K T A G Y charge (+6) 81 T F F Y G G K R G K R N N F K R E K Ycharge (+7) 82 T F F Y G G K R G K R N N F K R A K Y charge (0) 83 T F FY G G C L G N R N N F K T E E Y permut cys(−) 84 T F F Y G C G R G K R NN F K T E E Y permut cys(+) 85 T F F Y G G R C G K R N N F K T E E Ycharge (−4) 86 T F F Y G G C L G N G N N F D T E E E Q instead of F 87 TF Q Y G G C R G K R N N F K T E E Y ANGIOPEP 88 Y N K E F G T F N T K GC E R G Y R F scramble TFPI TFPI (similar 89 R F K Y G G C L G N M N N FE T L E E domain) Charge+5 90 R F K Y G G C L G N K N N F L R L K Y(HUMAN) Charge+5 91 R F K Y G G C L G N K N N Y L R L K Y (HUMAN) TFPI(c-terminal) 92 K T K R K R K K Q R V K I A Y E E I F K N Y (2Y) TFPI(c-terminal 93 K T K R K R K K Q R V K I A Y tronque) Basic- SynB1 94 RG G R L S Y S R R F S T S T G R Peptides SynB3 95 R R L S Y S R R R FPenetratin 96 R Q I K I W F Q N R R M K W K K (pAntp43-68)

TABLE 2B Reactive SEQ Peptide Peptide amines ID Name Sequences(positions) Charge No. Angiopep TFFYGGCRGKRNN 3 and 1 +2  67 1 FKTEEYcyst (1, 10, 15, 7) Angiopep- TFFYGGSRGKRNN 3 +2  97 2 FKTEEY(1, 10, 15) Angiopep- Ac¹-TFFYGGSRG 2 +1 107 3* KRNNFKTEEY (10, 15)Angiopep- RFFYGGSRGKR 3 +3 108 4b NNFKTEEY (1, 10, 15) Angiopep-Ac¹-RFFYGGSR 2 +2 109 4a GKRNNFKTEEY (10, 15) Angiopep- Ac¹-RFFYGGSR1 (10) +2 110 5 GKRNNFRTEEY Angiopep- TFFYGGSRGKR 2 +2 111 6 NNFRTEEY(1, 10) Angiopep- TFFYGGSRGRR 1 (1) +2 112 7 NNFRTEEY *Angiopep-3 is anacetylated form of Angiopep-2. ¹Ac represents acetylation.

It is to be understood herein, that if a “range” or “group ofsubstances” is mentioned with respect to a particular characteristic(e.g., temperature, concentration, time and the like) of the presentinvention, the present invention relates to and explicitly incorporatesherein each and every specific member and combination of sub-ranges orsub-groups therein whatsoever. Thus, any specified range or group is tobe understood as a shorthand way of referring to each and every memberof a range or group individually as well as each and every possiblesub-ranges or sub-groups encompassed therein; and similarly with respectto any sub-ranges or sub-groups therein. Thus, for example,

-   -   with respect to a length of 19 amino acid long or less, is to be        understood as specifically incorporating herein each and every        individual length, e.g., a length of 18, 17, 15, 10, 5, etc.;        Therefore, unless specifically mentioned, every range mentioned        herein is to be understood as being inclusive. For example, in        the expression from 5 to 19 amino acids long is to be as        including 5 and 19;    -   and similarly with respect to other parameters such as        sequences, length, concentrations, elements, etc. . . .

It is in particular to be understood herein that the sequences, regions,portions defined herein each include each and every individualsequences, regions, portions described thereby as well as each and everypossible sub-sequences, sub-regions, sub-portions whether suchsub-sequences, sub-regions, sub-portions is defined as positivelyincluding particular possibilities, as excluding particularpossibilities or a combination thereof; for example an exclusionarydefinition for a region may read as follows: “provided that saidpolypeptide is no shorter than 4, 5, 6, 7, 8 or 9 amino acids. Yet afurther example of a negative limitation is the following; a sequencecomprising SEQ ID NO.: X with the exclusion of a polypeptide of SEQ IDNO. Y.; etc. Other examples of exemplary negative limitations are thefollowing; “other than brain cancer” or “other than brain tissue” or“other than brain cells”.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrates exemplary embodiments of the invention,

FIG. 1. is an amino sequence of aprotinin-derived peptides;

FIG. 2. is a schematic representation of the efflux pump, P-glycoprotein(P-gp or MDR1) at the cell surface. The efflux pump, P-gp or MDR1,associated with multidrug resistance is highly expressed at the cellsurface of many cancer cells and various tissues including theblood-brain barrier;

FIG. 3A. is a schematic representation of conjugation of a drug to thecarrier peptide of the present invention;

FIG. 3B is a representation of the chemical structure of TxlAn2 (3:1);

FIG. 4. is a chromatogram illustrating the production in high amount ofTxlAn2 (3:1) conjugate;

FIG. 5. is a HPLC analysis of the peak purified on a hydrophobic columnusing AKTA-explorer;

FIG. 6. is a diagram illustrating the In situ brain perfusion ofradiolabeled Angiopep-2 and the vascular marker inulin;

FIG. 7. is an histogram illustrating the apparent distribution volume oftransferrin, aprotinin and Angiopeps in total brain, brain capillariesand brain parenchyma;

FIG. 8. is a diagram of cell proliferation in the presence of the parentdrug Taxol. Glioblastoma cells (U-87) were exposed to variousconcentrations of Taxol for 3-days. ³H-Thymidine incorporated in cellswere plotted as a function of Taxol concentrations;

FIG. 9A is a diagram representing the accumulation of various drugs inMDCK cells transfected with MDR1 in the presence or absence (control) of10 uM CsA, a P-gp inhibitor. The experiment was performed in thepresence of 1% DMSO;

FIG. 9B. is a diagram representing the accumulation of the conjugate incells over-expressing P-gp.

FIGS. 10A and 10B are diagrams representing tissue distribution of Taxoland TxlAn1 conjugate,

FIG. 11 is a diagram representing lung distribution of Taxol and TxlAn1,

FIG. 12 is a diagram representing the levels of TxlAn1 conjugate inplasma and lung,

FIG. 13A is a diagram representing the effect of TxlAn2 treatment onsubcutaneous glioblastomas (U-87) tumor growth;

FIG. 13B is a diagram representing the effect of high dose of TxlAn2(3:1) conjugate on SK-Hep 1 subcutaneous tumour growth;

FIG. 14. are photographs illustrating the detection of β-tubulin inNCI-H460 by immunofluoresence or visible light in cancer cells exposedto Taxol or TxlAn2 conjugate, as control, cells were exposed to 1% DMSO;

FIG. 15. are diagrams illustrating the effect of Taxol and TxlAn2conjugate on NCI-H460 cell-cycle measured by FACS. Cells were exposedfor 24 hrs with the vehicle (DMSO), Taxol (100 nM) or TxlAn2 conjugate(30 nM, equivalent to 100 nM of Taxol);

FIGS. 16A and 16B are pictures representing immunodetection of LRP inhuman brain tumor biopsies;

FIG. 17A. is a diagram illustrating the accumulation of [¹²⁵I]-RAP infibroblasts MEF-1 and PEA-13 in the presence of various aprotininconcentrations.

FIG. 17B. is a diagram expressing the results of FIG. 17A, where theLRP-dependent accumulation of [¹²⁵I]-RAP was calculated by subtractingthe results for the uptake obtained with PEA-13 cells from the resultswith MEF-1 and expressed as a function of aprotinin concentrations;

FIG. 18. is an histogram illustrating the effect of aprotinin andAngiopep-2 on RAP uptake. The accumulation of [¹²⁵I]-RAP in fibroblastsMEF-1 and PEA-13 was measured in the presence of 25 μM aprotinin orAngiopep. The LRP-dependent accumulation of [¹²⁵I]-RAP was calculated bysubtracted the results for the uptake obtained with PEA-13 cells fromthe results obtained with MEF-1;

FIG. 19A is a diagram illustrating the blood kinetics of TxlAn2 (3:1)conjugate in DMSO 80% after a bolus injection;

FIG. 19B is a diagram illustrating the blood kinetics of Tx1An2 (3:1)conjugate in macrogol 15 hydroxystearate (SOLUTOL® HS 15) 20% after abolus injection;

FIG. 19C is a diagram illustrating the blood kinetics of Taxol in DMSO80% after a bolus injection;

FIG. 20 are diagrams of tissue distribution or Tx1An2 (3:1) conjugatediluted in DMSO (80%) or macrogol 15 hydroxystearate (SOLUTOL® HS51)(20%);

FIG. 21A is an histogram illustrating tissue distribution of Taxol,TxlAn2 (3:1) and TxlAn2 (2:1) after intravenous (i.v.) injection inseveral tissues;

FIG. 21B is an histogram illustrating tissue distribution of Taxol,TxlAn2 (3:1) and TxlAn2 (2:1) after intravenous (i.v.) injection innormal and tumoral brain;

FIG. 22A is a graph illustrating tumor volume following i.v.administration of Taxol (10 mg/kg) or TxlAn2 (3:1) conjugate (20 mg/kg)formulation (in macrogol 15 hydroxystearate (SOLUTOL®) in mice withNCI-H460 cells implanted in their right flank;

FIG. 22B is a graph illustrating tumor volume following i.p.administration of Tx1An2 (2:1) or Tx1An2 (3:1) conjugate formulation(macrogol 15 hydroxystearate (SOLUTOL®)) in mice with NCI-H460 cellsimplanted in their right flank;

FIG. 23A is a graph illustrating tumor volume following administrationof either the vehicle (control), Taxol (10 mg/kg) or TxlAn2 (3:1)formulation (20 mg/kg) by i.p. injections or infusion with Alzet pumps(30 mg/kg/14 days) in mice with NCI-H460 cells implanted in their rightflank; Treatments are indicated by arrows, and;

FIG. 23B is a graph illustrating tumor volume following administrationof either the vehicle (control), Taxol (10 mg/kg) or TxlAn2 (3:1)formulation (20 mg/kg) by i.p. injections or infusion with Alzet pumpsin mice with U87 cells implanted in their right flank; Treatments areindicated by arrows.

DETAILED DESCRIPTION OF THE INVENTION

Angiopep-1 and -2 represents two non-limitative, exemplary embodimentsof aprotinin derived peptides which have been tested herein (FIG. 1).Taxol which represents a non-limitative exemplary embodiment of amolecule or compound conjugated to the carrier of the present inventionwas chosen as a candidate anticancer drug as this natural compound,isolated from the bark and needles of the yew tree, is a highlyefficient chemotherapeutic. Moreover, this compound is approved by theFood and Drug Administration (FDA) for ovarian cancer, breast cancer,non-small cell lung cancer and Kaposi's sarcoma and is a wellcharacterized anticancer agent.

EXAMPLES

Cell Proliferation Assay

For the in vitro cell proliferation assay, between 2.5 and 5×10⁴ of U87or A549 cells were seeded in a 24 well tissue culture microplate in afinal volume of 1 ml of medium with 10% serum and incubated for 24 hoursat 37° C. and 5% CO₂. The medium was then replaced with serum-freemedium and incubated overnight. The next morning the drug was freshlydissolved in dimethyl sulfoxide (DMSO) and the medium was replaced withcomplete medium containing the drug at different concentrations intriplicates. The final concentration of DMSO was 0.1%. The control usedis a microplate well with cells and without drug. The cells wereincubated for 48 to 72 hrs at 37° C. and 5% CO₂. After the incubation,the medium was changed and replaced with 1 ml of complete mediumcontaining [³H]-thymidine (1 μCi/assay). The plate was incubated at 37°C. and 5% CO₂ for 4 hrs. The medium was removed and the cells washedwith PBS heated at 37° C. The cells were fixed with a mix ofethanol:acetic acid (3:1), then washed with water and precipitated 3times with 10% of ice-cold TCA (trichloroacetic acid). Finally 500 μl ofPCA (perchloric acid) were added to the wells and the microplates wereheated for 30 min at 65° C. and 30 min at 75° C. The contents of eachwell was then transferred in a scintillation vial with 10 ml ofscintillation cocktail and the activity was measured in CPM (count perminute) on a liquid scintillation counter Tri-Carb from Packard.

Iodination of Peptides

Peptides were iodinated with standard procedures using iodo-beads fromSigma. Both Angiopep-1 and Angiopep-2 were diluted in 0.1M phosphatebuffer, pH 6.5 (PB). Two iodo-beads were used for each protein. Thesebeads were washed twice with 3 ml of PB on a whatman filter andre-suspended in 60 μl of PB. ¹²⁵I (1 mCi) from Amersham-Pharmaciabiotech was added to the bead suspension for 5 min at room temperature.Each iodination was initiated by the addition of the peptide (100 μg).After an incubation of 10 min at room temperature, the free iodine wasremoved by HPLC.

Subcutaneous Implantation

In order to estimate the efficiency of the Taxol-conjugates andformulations on tumor growth, we developed a subcutaneous model ofglioblastomas. In this model, 2.5×10⁶ cells in 100 μl of cell mediumwithout serum containing 1% methylcellulose were subcutaneously injectedin the mice flank. The tumor was clearly visible and could be measuredusing a vernier caliper. The estimated tumor volume was then plotted asa function of time.

In Situ Mouse Brain Perfusion

The uptake of [¹²⁵I]-peptides to the luminal side of mouse braincapillaries was measured using the in situ brain perfusion methodadapted in our laboratory for the study of drug uptake in the mousebrain. Briefly, the right common carotid of ketamine/xylazine (140/8mg/kg i.p.) anesthetized mice was exposed and ligated at the level ofthe bifurcation of the common carotid, rostral to the occipital artery.The common carotid was then catheterized rostrally with polyethylenetubing filled with heparin (25 U/ml) and mounted on a 26-gauge needle.The syringe containing the perfusion fluid ([¹²⁵I]-peptides or[¹⁴C]-inulin in Krebs/bicarbonate buffer at a pH7.4 gassed with 95% O₂and 5% CO₂) was placed in an infusion pump (Harvard pump PHD 2000;Harvard Apparatus) and connected to the catheter. Prior to theperfusion, the contralateral blood flow contribution was eliminated bysevering heart ventricles. The brain was perfused for the indicatedtimes at a flow rate of 1.15 ml/min. After 14.5 min of perfusion, thebrain was further perfused for 60 s with Krebs buffer, to wash theexcess of [¹²⁵I]-proteins. Mice were then decapitated to terminateperfusion and the right hemisphere was isolated on ice before beingsubjected to capillary depletion. Aliquots of homogenates, supematants,pellets and perfusates were taken to measure their contents in[¹²⁵I]-conjugates by TCA precipitation and to evaluate the apparentvolume of distribution.

Example 1—Preparation of Conjugates

Since the resistance towards various drugs such as vincristine,etoposide, and doxorubicin is mediated through P-gp (MDR1)overexpression (FIG. 2), any methods of bypassing this efflux pump maypotentiate the action of these drugs on various cancer types. The bypassof P-gp may therefore be useful to increase the potency of drugs whichare associated with resistance mediated by P-gp. Carriers describedherein were therefore tested for their ability to bypass P-gp.

The conjugation of a drug to the carrier described herein is illustratedin FIG. 3. Briefly, in order to conjugate Taxol to the Angiopep-1 orAngiopep-2 carrier, Taxol was first activated into N-succinimide(2′-NHS-Txl) derivative. Then amines found for example in lysine residueor amino-terminal of Angiopep-1 or Angiopep-2 reacted on thisactivated-Taxol by forming a peptide bond (amide bond). In Angiopep-1 orAngiopep-2, the amino-terminal (in position 1) and the lysines (atposition 10 and 15) were able to react with activated-Taxol. Therefore,multiple combinations of conjugates was found to occur by the additionof 1, 2 or 3 Taxols to the peptide depending on the molar ratio used(FIG. 3). The whole conjugation was analyzed by HPLC and conjugation wasconfirmed by Mass spectra (Maldi-Tof). Taxol was found to be releasablefrom the carrier by the cleavage of the ester bond by esterases.Conjugates were therefore efficiently produced by combining the carrierwith an anticancer drug.

In an exemplary embodiment of the present invention, the production ofthe TxlAn2 (3:1) conjugate, was carried out by directly adding 1 moleequivalent of Angiopep-2 to a solution of 2.5 moles equivalent of2′-NHS-Taxol. The reaction was performed in 68% DMSO in Ringer solution(pH 7.3), for 1 hr at 12° C. and after removing the cold bath, for about22 hrs at room temperature (FIG. 4). Angiopep-2, 2′-NHS-Taxol and TxlAn2(3:1) conjugate are shown on the chromatogram by arrows. Aliquots of thereaction were sampled and analyzed by HPLC after 25 min, 2 hrs 15 min, 5hrs and 23 hrs as indicated in FIG. 4. The peaks of Angiopep-2,2′-NHS-Taxol and TxlAn2 (3:1) conjugate are shown by arrow on thechromatogram. Results of FIG. 4 illustrate the disappearance ofAngiopep-2 and 2′-NHS-Taxol during the reaction mainly to the profit ofthe TxlAn2 (3:1) conjugate.

This mixture of products was separated by hydrophobic chromatography ona RPC 300 mm column with a flow rate at 4 ml/min using AKTA-explorer(FIG. 5). For the peak that corresponds to the TxlAn2 (3:1) conjugate,fractions were pooled, analyzed by HPLC and MS. In FIG. 5, the upperchromatogram corresponds to the running reaction at t=23 hrs whereas thelower one corresponds to the TxlAn2 (3:1) conjugate which has beenconfirmed by mass spectrometry (MW 5107) after AKTA purification.

Example 2—In Situ Brain Perfusion of Taxol-Angiopep-2 Conjugates

To evaluate the brain uptake of Angiopep in vivo, the initial rate oftransport for [¹²⁵I]-Angiopep into mouse brain parenchyma was measuredusing in situ brain perfusion as described herein. Mouse brain wasperfused for the indicated times with either [¹²⁵I]-Angiopep-2 or[¹⁴C]-inulin. After perfusion, the brain was further perfused for 60 secwith Ringer solution to wash the excess of radiolabeled molecules andthen the right hemisphere was isolated on ice before being subjected tocapillary depletion. Aliquots of homogenates, supernatants, pellets andperfusates were taken to measure their contents in [¹²⁵I]-Angiopep-2 or[¹⁴C]-inulin. Results obtained for the accumulation for these moleculesinto the brain parenchyma are illustrated in FIG. 6. The accumulation of[¹²⁵I]-Angiopep-2 increased as a function of time and is higher thanthat of the vascular marker, [¹⁴C]-inulin.

We further compared the initial brain uptake after a 5 min perfusion for[¹²⁵I]-aprotinin, [¹²⁵I]-transferrin and [¹²⁵I]-Angiopeps (FIG. 7)(1:1). Results show that Angiopep and aprotinin have a highest initialtransport rate than transferrin.

Example 3—Effect of Conjugates on Cell Growth

In an in vitro assay, Taxol (unconjugated) was shown herein to block theproliferation of glioblastoma cells (U-87) with IC50 value of around 10nM (FIG. 8). The effect of Taxol conjugated with the carrier describedherein on the proliferation of various cell lines was then evaluated andcompared to unconjugated Taxol (referred as Taxol). As shown in Table3A, the IC50 values obtained for the Taxol-Angiopep-2 (TxlAn2) conjugatewere very similar to that of unconjugated Taxol in many cancer cells.Endothelial cells from rat brain (RBE4) were less sensitive than thetested cancer cell lines. Overall, these results indicate that thepotency of conjugates to block cell proliferation in vitro is similar tothe unconjugated Taxol. For comparison purposes, results obtained wereexpressed in term of Taxol concentration.

TABLE 3A Effect of conjugate on cell proliferation. IC50 (nM) Cell linesTaxol Taxol-Angiopep-2 (3:1) Glioblastomas U-87 9.5 9.7 U-118 7.2 8.1Lung carcinoma NCI-H460 9.3 12.5 A549 3.6 6.0 Calu-3 17.2 25.0Endothelial cells RBE4 137 139 Most of theses cells (U-87, U-118,NCI-H460, A549) express LRP. This data is however unavailable for RBE4cells.

The anti-proliferatice activity of the conjugate against cancer cells invitro was further assessed. In this assay, cancer cells (U87 and U118)were exposed for 48 hrs to Taxol® and TxlAn2 (3:1) conjugate.Incorporation of [³H]-thymidine in U87 and U118 cells decreased as afunction of drug concentrations. The values required to inhibit cellproliferation by 50% (IC50) were expressed. Results obtained from theproliferation assays indicate that the IC50 values required for theinhibition of cancer cell proliferation are expressed in nM anddemonstrate that TxlAn2 (3:1) conjugate is 3 times more potent thanpaclitaxel, and are in the same range when reported in paclitaxelequivalent (Table 3B).

The capacity of TxlAn2 (3:1) conjugate to block the proliferation ofother cancer cell types was also estimated. Lung cancer cells (NCI-H460)as well as the breast cancer cell line (MDA-MB231, MDA-MB-468, HCC-1954,BT-474) in hepatocarcinomas (SK-Hep1) and glioblastomas (U-87MG) werealso very sensitive to TxlAn2 (3:1) conjugate (Table 3B).

TABLE 3B In vitro cytotoxicity of taxol and TxlAn2 (3:1) conjugatePaclitaxel TxlAn2 (3:1) conjugate Residual Residual IC50 (nM) survival %IC50 (nM) survival % Cell line Mean SD Mean SD Mean SD Mean SD BT-47462.86 — 52.24 3.87 40.22 — 51.83 1.70 HCC1954 6.12 — 48.82 11.17 8.26 3.37 44.08 8.63 MDA-MB-231 17.61 — 45.62 13.89 28.16 — 46.95 7.50MDA-MB-468 13.52 — 54.26 10.34 1.41 — 55.34 5.55 NCI-H460 6.61 3.3524.98 10.80 12.68 10.08 30.44 9.36 SK-HEP-1 8.84 — 55.18 13.17 7.83 —54.09 11.03 U-87 MG 12.94 2.49 40.35 2.40 17.75 10.82 44.13 1.85

Example 4—By-Passing of P-gp by Conjugates

In order to determine whether the conjugates of the present inventionwere P-gp substrates or not, MDCK cells were stably transfected withhuman MDR1 (MDCK-MDR1) and were subsequently incubated withunconjugated-anticancer drug or with the conjugates of the presentinvention. In a first experiment, MDCK-MDR1 cells were incubated with³H-vinblastine (³H-VBL), rhodamine, ³H-Taxol, ¹²⁵I-Taxol-Angiopep-1(¹²⁵I-TxlAn1), ¹²⁵I-Taxol-Angiopep-2 (¹²⁵I-TxlAn2) for 1 hr at 37° C. inthe presence or absence of 10 μM of cyclosporine A (CsA); a P-gpcompetitive inhibitor (FIG. 9A). After the incubation, cells were washedand accumulation of radioactivity inside the cells or intracellularfluorescence was quantified. The increased in the accumulation of thesedrugs is expressed in term of x-fold compared to their respectivecontrol measured in the absence of CsA. Thus, the control value for eachdrug was set to 1-fold.

In another experiment, the ability of the conjugates to accumulate incells overexpressing P-gp was monitored (FIG. 9B). MDCK-MDR1 cells wereincubated with 50 nM of either ³H-Taxol, ¹²⁵I-Taxol-Angiopep-1(¹²⁵I-TxlAn1) or ¹²⁵I-Taxol-Angiopep-2 (¹²⁵I-TxlAn2) for 2 hrs at 37° C.After the incubation, the cells were washed and the radioactivityaccumulated in cells was quantified. The results were expressed as drugaccumulation in pmole/120 min.

As shown in FIG. 9A, the accumulation of [³H]-Taxol increased by 15-foldin the presence of the P-gp competitive inhibitor; cyclosporin A (CsA).The accumulation of rhodamine and [³H]-vinblastine also increased by7.5-fold and 10-fold respectively in the presence of CsA. These resultsshow that Taxol, rhodamine and vinblastine are P-gp substrates. However,the lack of CsA effect on the accumulation of both[¹²⁵I]-Taxol-Angiopep-1 and [¹²⁵I]-Taxol-Angiopep-2 conjugates,indicates that they are not P-gp substrates. The accumulation of bothconjugates, in the absence of CsA, was at least 11-fold higher than of[³H]-Taxol (FIG. 9B). These later results strongly confirm that bothconjugates bypass the action of P-gp since P-gp is not able to expulsethem from the cells. These results additionally demonstrate that thepresence of a carrier in conjugation with an anticancer drug increasesthe potency of the drug. Therefore, the carriers described herein areuseful for the transport and/or accumulation of drugs inside a cell andare especially useful for drugs which are usually expulsed by P-gp(i.e., drugs which are P-gp substrates).

Example 5—Distribution and Pharmacokinetics of Conjugates

The impact of conjugation of the drug to the carrier on drugdistribution was evaluated by administering either ³H-Taxol (5 mg/kg) or¹²⁵I-Taxol-Angiopep-1 (TxlAn-1) (10 mg/kg, equivalent to 5 mg ofTaxol/kg) to mice. The unconjugated anticancer drug and the conjugateswere injected intra-venously (i.v.) in mice as a bolus. Tissues werecollected at different times (0.25, 0.5, 1 and 4 hrs) and homogenized.In order to quantify the amount of ³H-Taxol, tissue homogenates weredigested with tissue solubilizer (soluble) and 10 ml of liquidscintillator was then added to samples. The amount of the ¹²⁵I-labeledconjugate, in the different tissues was measured after TCAprecipitation. Radioactivity associated with the tissues was thereforequantified. The area under the curve (AUCO-4) was estimated using thePrism software and was plotted for the different tissues (FIG. 10).Results of FIG. 10A indicate that the AUCO-4 values obtained for theconjugate are higher than that of Taxol in various tissues including thebrain, kidney, liver and the eyes which indicates a higher accumulationof the conjugate in these tissues compared to the unconjugated drug.More particularly, results presented in FIG. 10B indicate that theaccumulation of the conjugate is much higher than unconjugated drug inthe lung.

Results of a similar experimentation conducted with the Taxol-angiopep-2conjugate are summarized in Table 4 below. Although there is differencewith results obtained for the TxlAn-1 conjugate, the conjugate of Table4 also accumulates in the lungs, brain and liver more efficiently thanunconjugated Taxol.

TABLE 4 AUC 0-4 (μg/g of tissue) Tissue TxlAn-2 Taxol Ratio(TxlAn-2/Taxol) Plasma 170 2.2 77.3 Brain 0.32 0.07 4.6 Lung 3.4 1.1 3.0Kidney 11.2 8.0 1.4 Heart 5.0 2.5 2.0 Liver 513 22 23 Eye 0.99 0.57 1.7Urine 35.7 88 0.4 Treatments equivalent to 5 mg/kg of Taxol

The kinetics of Taxol and Taxol-Angiopep-1 accumulation in the lung isalso presented in FIG. 11. Results clearly show that the amount of theconjugate measured in the lungs at different times is much higher thanfor the unconjugated drug. Moreover, we also observed that theaccumulation of the conjugate in the lung is also much higher than itsconcentration in the serum (plasma) at various times (FIG. 12). Resultspresented in FIGS. 10, 11 and 12, strongly indicate that the conjugationof an anticancer drug (e.g., Taxol) to the carrier of the presentinvention (e.g., Angiopep-1 or 2) modifies the biodistribution of theanticancer drug and its pharmacokinetics.

Example 6—Inhibition of Tumor Growth In Vivo (U-87)

The ability of conjugate to inhibit tumor growth was next evaluated inan in vivo model (FIG. 13A). U-87 cells were therefore subcutaneouslyimplanted in the right flank of mice and on day 3 post-implantation,mice were injected with the vehicle (DMSO/Ringer: 80/20; control), Taxol(5 mg/kg) or Taxol-Angiopep-2 (10 mg/kg; equivalent to 5 mg of Taxol/kg(3 Taxol: 1 Angiopep-2)). We observed that the tumor growth inhibitionwas more pronounced in mice treated with the conjugate than in micetreated with the unconjugated anticancer drug.

In fact at day 17 post-implantation, tumor growth was inhibited by morethan 75% by the conjugate whereas tumor growth was inhibited by only 34%using the unconjugated drug (Table 5). These results show that theconjugates described herein are more efficient than unconjugated Taxolat inhibiting tumor growth in vivo. Overall, a 2.2-fold tumor growthinhibition level was measured for the conjugate compared to theunconjugated drug.

TABLE 5 Inhibition of tumor growth with conjugates. Tumor volume (mm³)(mean ± sem) Tumor Days post-injection growth Inhibition T/C Groups Day0* Day 14** Δ (mm³) (%) (%) Control 79 ± 7 289 ± 50 203 ± 47 100 Taxol74 ± 5 219 ± 52 134 ± 55 34 66 (5 mg/kg) TxlAn2 (3:1) 88 ± 9 144 ± 27 56 ± 32 73 27 (10 mg/kg) Treatment equivalent to 5 mg/kg of Taxol *corresponds to 3 days post-implantation (first treatment)**corresponds to 17 days post-implantation (after 4 treatments)

Example 7—Inhibition of Tumor Growth In Vivo (Hepatocarcinoma)

In vivo studies were conducted to determine whether TxlAn2 (3:1)conjugate could inhibit the growth of Hepatocarcinoma cells (SK-Hep 1)that have been implanted subcutaneously. For these in vivo models, nudemice received a subcutaneous injection of 2.5×10⁶ human SK-Hep 1 cellsin their right flank. Different treatments were started once the size ofthe implanted tumor reach approx. 200 mm³. Animals received treatmentTxlAn2 (3:1) conjugate or vehicle by peritoneal injections (i.p.).TxlAn2 (3:1) conjugate was administered at 80 mg/kg by IP injection(TxlAn2 (3:1) conjugate was taken from 2 different batch stored indifferent conditions). In FIG. 13B treatments are indicated by blackarrows.

Treatments were given twice a week for 5 treatments maximum at the doseindicated of 80 mg/kg. FIG. 13B shows that TxlAn2 (3:1) conjugate whenadministered i.p., shows high efficacy in inhibiting the growth ofhepatocarcinomas. This type of cancer is usually not sensitive toTaxol®.

Example 8—Mechanism of Action of Conjugates

In FIG. 14, lung cancer cells (NCI-H460) were incubated for 24 hrs witheither free Taxol (30 nM) or TxlAn2 conjugate (10 nM; equivalent to 30nM of Taxol). After cells were labeled for β-tubulin by using asecondary antibody linked to FITC. Pictures were taken in visible andfluorescence modes. These results indicate that both Taxol andTaxol-Angiopep conjugate have similar effect on β-tubulin leading to itspolymerization. Moreover, as indicated in FIG. 15, the addition of Taxoland Taxol-Angiopep conjugate induce a blockade of NCI-H460 cell in G2/Mphase. Results obtained for the β-tubulin polymerization and cell cyclesuggest that TxlAn conjugate has a similar mechanism of action on cancercells than Taxol.

Example 9—Effect on LRP-Mediated RAP Accumulation

It was previously shown in international patent application No.PCT/CA2004/00011, that the receptor-associated protein (RAP) inhibitedtranscytosis of aprotinin in an in vitro model of the blood brainbarrier. According to these data we proposed that the low-densitylipoprotein related receptor (LRP) is involved in the penetration ofaprotinin into the brain. Similar inhibition of Angiopep transportacross an in vitro model of the blood-brain barrier was also obtained(data not show) suggesting that transcytosis of Angiopep across brainendothelial cell also involved LRP. LRP is a heterodimeric membranereceptor of 600 kDa composed of two subunits; the subunit-α (515 kDa)and the subunitβ (85 kDa). Immunodetection of LRP was then performed toassess whether this receptor is expressed in human primary brain tumorssuch as glioblastomas and in human brain metastasis from breast, lungand melanoma cancers (FIG. 16). Briefly, equal amount of proteinhomogenates from human primary brain tumors (glioblastomas) or humanbrain metastasis were separated by gel electrophoresis. Afterelectrophoresis, proteins were transferred to PVDF membrane and LRP wasimmunodetected by using a monoclonal antibody directed against thesubunit-α obtained from Cedarlane Laboratories (Hornby, ON, Canada). LRPwas visualized by a secondary antibody directed against mouse IgG linkedto horseradish peroxidase and chemiluminescence reagents.

Under the experimental conditions used, the subunit a of LRP wasimmunodetected at 515 kDa in glioblastoma U-87 cells. LRP was alsodetected in all human primary brain tumors and human brain metastases(FIG. 16). In contrast, megalin (LRP2) was detected in only one brainmetastasis of the lung (not shown). The expression of LRP in thedifferent patient biopsies may explain in part why we previouslyobserved a higher accumulation of Taxol-Aprotinin conjugate in braintumors. Overall, since LRP may be involved in the transport of thecarrier described herein, these results indicate that the conjugates mayalso target cells and tumors which express this receptor.

In order to determine whether aprotinin and Angiopep transcytosis couldinvolve LRP, their impact on the uptake of the receptor-associatedprotein (RAP), an endogenous ligand for LRP, was determined (FIG. 17A).The uptake of RAP was measured in fibroblasts expressing LRP (MEF-1) andin fibroblasts that do not express LRP (PEA-13) (FIGS. 17A and 17B). Theaddition of aprotinin inhibited the transport of [¹²⁵I]-RAP in positiveLRP cells in a dose-dependent manner. In contrast, the RAP uptake innegative LRP cells was almost unaffected by these aprotininconcentrations. In FIG. 17B the difference between the uptake of[¹²⁵I]-RAP measured in MEF-1 and PEA-13 was calculated and plotted as afunction of aprotinin concentration. These results show that a portionof the LRP-dependent uptake of RAP could be reduced by aprotininindicating that aprotinin could interact with this receptor. In adifferent experiment (FIG. 18), the uptake of [¹²⁵I]-RAP was alsomeasured in the presence of an excess of aprotinin and Angiopep. Resultsshow that both aprotinin and Angiopep affect the LRP-dependentaccumulation of [¹²⁵I]-RAP.

In summary, data obtained for the conjugates described herein indicatethat the conjugation of anticancer drugs to the carrier allows theanticancer drug to escape from P-gp action and therefore increase theirpotency (when conjugated with the carrier). These conjugates are activein vitro at inhibiting cancer cell proliferation. Moreover, resultsobtained on in vivo tumor growth indicate that the conjugation ofanticancer drug to the carrier may increase their efficiency bybypassing P-gp, possibly targeting the receptor LRP or by modifying thepharmokinetics or biodisponibility of the unconjugated drug.

Taken together, data described herein indicates that the conjugates maybe used against primary tumors including breast, lung and skin cancersas wells as metastasis originating from primary tumors.

Example 10—Improved Formulation of Taxol-Angiopep Conjugates

Preliminary assays performed to assess the solubility of the differentTxlAn conjugates indicated that all conjugates had a low solubility inaqueous solution (e.g., in Ringer/Hepes solution) due to the highlyhydrophobic nature of Taxol. However, all of the conjugates were verysoluble in dimethyl sulfoxide (DMSO)/Ringer (80%/20%). Differentstrategies were thus assessed to increase their solubility and to reducethe amount of DMSO necessary for their solubilization. Interestingly, wewere able to completely remove DMSO from the formulation by using thesolubilizer agent macrogol 15 hydroxystearate (Solutol® HS15 (BASF)).For example, TxlAn2 (3.1) at 5 mg/ml was efficiently solubilized in 20%macrogol 15 hydroxystearate (Solutol® HS15) and Ringer/Hepes solution pH5.5. As this agent has been approved for several drugs applications forintravenous (i.v.) and intraperitoneal (i.p.) administration, its useprovides a commercial advantage to the formulations of the presentinvention.

Formulations of the present invention may thus comprise, for example, a)Taxol-Angiopep conjugates, b) macrogol 15 hydroxystearate (Solutol®HS15) and c) an aqueous solution or buffer (e.g., Ringer/Hepes solutionat a pH of 5 to 7). The concentration of macrogol 15 hydroxystearate(Solutol® HS15) in the formulation may reach, for example, 30%.Concentration higher than 30% may also be useful. The concentration ofconjugate may be determined based upon the dose required for efficientlytreating a patient.

Example 11—Blood Kinetics of the Improved Formulations

For tissue distribution and blood kinetic studies, iodinated[¹²⁵I]-conjugates (i.e., [¹²⁵I]-TxlAn conjugates) and [³H]-Taxol wereused. Briefly, TxlAn2 conjugates (1 mg) were radioiodinated usingiodobeads and Txl-[¹²⁵I]-An2. Conjugate was then purified using a columncontaining resource RPC resin. Free iodine was removed by washing thecolumn thoroughly with 20% acetonitrile. During column washesradioactivity was counted to assess the decrease in free iodine. TheTxl-[¹²⁵I]-An2 conjugate was then pulled-down by a 100% acetonitrilewash. Acetonitrile was then evaporated and the iodinated conjugate wasdiluted in 100% DMSO (100 μL). An aliquot of the radioiodinatedconjugate was then injected in HPLC and fractions were collected toverify that the radioactivity was associated to the fractionscorresponding to the conjugates.

Blood kinetics were assessed after intravenous ((i.v.) tail vein),intraperitoneal (i.p.) and subcutaneous (s.c.) injections performed onawake mice (FIG. 19) Briefly TxlAn2 (3:1) conjugate was diluted inDMSO/Ringer-Hepes (80/20) or in macrogol 15 hydroxystearate(Solutol®)/Ringer-Hepes (20/80), Txl-[¹²⁵I]-An2. Injections of CD-1 micewith the formulations were then performed to obtain a 10 mg/kgconcentration. After injections blood fractions (50 μL) were collectedat the tail end and radioactivity was directly assessed. Using the sameprotocol, Taxol blood kinetic was also determined using [³H]-Taxol.Taxol was dissolved in DMSO/Ringer-Hepes (80/20) at a concentrationallowing a 5 mg/kg injection and [³H]-Taxol was then added (2.5μCi/injection). After injections blood fractions were collected at thetail end, scintillation cocktail was added and radioactivity was countedin a Packard counter. Results of this experiment are illustrated in FIG.19A, FIG. 19 and in FIG. 19C and are summarized in Table 6 below.

In summary, results of FIG. 19 and Table 6 show that TxlAn2bioavailability is much higher than Taxol bioavailability. For example,the AUC_((0-24hrs)) TxlAn2/AUC_((0-24hrs)) Taxol is 169 (i.e.,203.3/1.2).

In terms of Taxol, the AUC_((0-24hrs)) TxlAn2/AUC_((0-24hrs)) Taxol is84.7 (i.e., 101.65/1.2).

Since there is three Taxol molecules on each molecule of Angiopep-2, theamount of Taxol represents about 0.5 of Angiopep's molecular weight(i.e., 3×854/5301). Therefore the AUC of the conjugate (i.e., 203) hasto be multiplied by 0.5 in order to be expressed in term of Taxol.

In addition the blood biodisponibility of TxlAn2 conjugates isequivalent after intravenous and intraperitoneal injections whereas thisis not the case for Taxol. Finally, results of FIG. 19 and Table 6indicate that the blood biodisponibility of TxlAn2 is higher whenmacrogol 15 hydroxystearate (Solutol®) is used as solubilizer comparedto DMSO.

TABLE 6 Area under curves from 0 to 24 hours were calculated usingGraphPad software. AUC (0-24 h) Intravenous Intraperitoneal Subcutaneous(μg · h per mL) injection injection injection TxlAn2 (3:1) (DMSO) 98.134.7 15.5 (10 mg/kg) TxlAn2 (3:1) 203.3 211.3 74.5 (Macrogol 15hydroxystearate (Solutol ®) (10 mg/kg) Taxol 1.2 0.09 0.14 (DMSO) (5mg/kg)

Example 12—Tissue Distribution

TxlAn2 tissue distribution was evaluated in normal CD-1 mice after tailvein intravenous injection of 10 mg/kg TxlAn2 solubilized in macrogol 15hydroxystearate (Solutol®)/Ringer-Hepes (20%/80%) or inDMSO/Ringer-Hepes (80/20). Briefly CD-1 mice were injected via the tailvein with a formulation of TxlAn2 solubilized in macrogol 15hydroxystearate (Solutol®) or DMSO and also containing Txl-[¹²⁵I]-An2.At predetermined time points a blood sample was collected andanesthetized mice were perfused with cold PBS. Tissues were then excisedand radioactivity was counted in a gamma counter.

Results of FIG. 20 show that the use of macrogol 15 hydroxystearate(Solutol®) allows a higher distribution of TxlAn2 (3:1) conjugate inmost tissues.

Example 13—Preliminary Comparative Toxicity of Paclitaxel and TxlAn2(3:1) Conjugate

A direct comparison of the toxicity of paclitaxel versus TxlAn2 (3:1)conjugate was made on beagle dogs, using IV injection of 2.5 mg/kg ofpaclitaxel vs 5 mg/kg of TxlAn2 (3:1) conjugate. Four dogs were treatedin each cohort. The following observation was made:

Preclinical Observation

During initial infusion, paclitaxel was not well tolerated, as opposedto TxlAn2 (3:1) conjugate which was very well tolerated.

Later on, the paclitaxel group lost weight during days 2 and 3 andrecovered. No significant weight loss for TxlAn2 (3:1) conjugate.

Biological Observation

TABLE 7 Clinical pathology White Blood Cells Reticulocytes Platelets×109 cells/L ×1012 cells/L ×109 cells/L TxlAn2 TxlAn2 TxlAn2 (3:1) (3:1)(3:1) Taxol ® conjugate Taxol ® conjugate Taxol ® conjugatePre-treatment 9.7 10.6 0.031 0.044 250 275 4-days post- 4.3 9.7 0.0110.065 200 275 treatment 8-days post 8.7 10.4 0.11 0.10 340 290 treatment

These biological observations demonstrate that at an equivalentmolecular dose of Taxol®, TxlAn2 (3:1) conjugate does not trigger bonemarrow toxicity as opposed to paclitaxel alone. The more favorabletoxicity profile than paclitaxel will allow administration of TxlAn2(3:1) conjugate at a higher dosage than Taxol®, thus further increasingthe concentration of active drug to the tumor.

Example 14—Toxicity Studies

Single Acute Dose Toxicity in Rats and Dogs (GLP)

Single infusions of 0, 100, 200, 400, 850 mg/m² (14-120 mg/kg of TxlAn2(3:1) conjugate) in rats (n=3/sex/group):

The observations are as fellows:

-   -   Dose-dependent hematological effects (decreased platelets, WBCs        and reticulocytes) observed at all dose levels—maximum effects        on Day 4 with recovery thereafter    -   Some decreases in body weight gain (10-15%) at 850 mg/m2    -   Clinical chemistry normal, no remarkable macroscopic findings    -   Maximum Tolerated Dose (MTD) determined at 400 mg/m² or 56 mg/kg

Single infusions of 0, 100, 200, 400 mg/m² (5-20 mg/kg of TxlAn2 (3:1)conjugate) in dogs (n=1/sex/group):

The observations are as fellows:

-   -   Transient anaphylactoid reactions (face and head swelling) to        macrogol 15 hydroxystearate (Solutol®)    -   Dose-dependent hematological effects    -   High dose male and female dogs sacrificed on Day 4    -   Mid dose not well tolerated by female (poor food consumption for        ˜1 week)    -   MTD determined to be at 200 mg/m² or 20 mg/kg        Dose Range Finding Toxicity Studies in Rats and Dogs (Non-GLP)        Twice weekly infusions of 0, 25, 75, 150 mg/m² for 2 weeks (4        doses total) in rats (n=3/sex/group):

The observations are as follows:

-   -   Dose-dependent hematological effects (platelets, WBCs,        reticulocytes, Hb)    -   1 high dose female found dead on Day 10 (2 days post 3rd dose);        1 high dose male sacrificed on Day 15 (4 days post 4th        dose)—both rats had low hematological values    -   Infusion twice a week at 75 mg/m² is well tolerated for 2 weeks.        Twice weekly infusions of 0, 25, 75, 150 mg/m² for 2 weeks (4        doses total) in dogs (n=1/sex/group):

The observations are as follows:

-   -   Dose-dependent hematological effects (platelets, WBCs,        reticulocytes, Hb)    -   High dose female found dead on Day 7 and high dose male        sacrificed on Day 7 (poor food consumption and body        deterioration)    -   Infusion twice a week at 75 mg/m² is well tolerated for 2 weeks.        TxlAn2 (3:1) conjugate is well tolerated and as indicated in        Table 8 is better tolerated than Paclitaxel or Abraxane.

TABLE 8 MTD comparison with Paclitaxel: data based on rat GLP toxicityprograms Improvement compared Product MTD to Paclitaxel Paclitaxel  8mg/kg 3.5 times TxlAn2 (3:1) conjugate 56 mg/kg (28 mg/kg) Abraxane(ABI-007) 12 mg/kg 1.5 times Abraxis Inc. Paclitaxel incorporated andbound to human serum albumin associated to nanoparticlesTocosol-Paclitaxel (TP) 12 mg/kg 1.5 times Sonus Pharmaceutical Inc.Vitamin E based Taxol Emulsion incorporating a Pgp inhibitor andparticle size-based tumour Xyotax (PG-TXL) 16 mg/kg   2 times CellTherapeutics Inc. Paclitaxel polyglumex, biodegradable polyglutamatepolymer of paclitaxel

Example 15—Distribution in Brain Tumors

In an attempt to evaluate the distribution of TxlAn2 (3:1) conjugate ina brain tumor model, nude mice were intracerebrally implanted withNCI-H460 lung cancer cells. Ten days after implantation, mice weightloss was significant indicating that the brain tumors were wellestablished. Taxol, TxlAn2 (3:1) and TxlAn2 (2:1) tissue distributionswere evaluated (FIG. 21), as precedently described.

Mice were thus given an intravenous injection of either Taxol (5 mg/kg)solubilized in DMSO or TxlAn2 (3:1) (10 mg/kg) or TxlAn2 (2:1) (12.5mg/kg) each solubilized in macrogol 15 hydroxystearate (Solutol®). After10 minutes, mice were perfused on ice using cold PBS, organs werecollected and radioactivity was measured. To evaluate the difference ofaccumulation between normal brain and brain tumor, brains were cut inhalf with the right hemisphere (site of injection of the tumor cells)corresponding to the tumor brain and the left hemisphere to the normalbrain.

Results of FIG. 21A and FIG. 21B show that TxlAn2 (3:1) conjugatespresent a higher distribution in brain tumor compared to normal brain(2-fold increase) whereas no difference is observed for Taxoldistribution between normal and tumoral brain. TxlAn2 (3:1) conjugatedistribution is much higher than Taxol distribution in brain tumor(10-fold increase) and was also higher than TxlAn2 (2:1) distribution(4.5-fold).

Example 16 Effect of Taxol-Angiopep Conjugates Improved Formulation ons.c. Tumor Growth

In vivo studies were conducted to determine whether the improvedformulation comprising the Taxol-Angiopep conjugate could inhibit lungcancer cell (NCI-H460) growth or glioblastoma cells (U87) growth in anin vivo model of mice implanted subcutaneously with these cancer cells.

Briefly, mice received a subcutaneous injection of 2.5×10⁶ human U87glioma cells or NCI-H460 cells. When tumor growth was observed, micereceived treatment with free Taxol, Taxol-Angiopep conjugates or vehicleby i.v. or i.p. injections. Treatments were then administered twice aweek until animals were sacrificed. Mice were monitored every day forclinical symptoms and weight loss. Tumor volume was estimated with akaliper and the following equation (tumor volume=π/2×(length (mm)×width²(mm)).

In the first subcutaneous tumor growth study, NCI-H460 cells wereimplanted in mice right flank (FIG. 22A). Mice received the vehicle,Taxol or Taxol-Angiopep-2 (3:1) conjugate formulation by i.v. injectionsin the tail vein or i.p. injections. Conjugates were administered at anequivalent of 10 mg/kg of Taxol. Results presented in FIG. 22 show thatthe improved formulation of TxlAn2 (3:1) conjugate containing 20%macrogol 15 hydroxystearate (Solutol® HS15) in Ringer/Hepes solution (pH5.5) caused a much stronger inhibition of NCI-H460 tumor growth thanTaxol. These results are also summarized in Table 9 below.

TABLE 9 Tumor volume (mm³) Tumor Days post-injection growth T/CMolecules Day 0 Day 11 Δ (mm³) (%) Macrogol 15 118 ± 11 714 ± 71 596 ±62 100 hydroxystearate (Solutol® ) Taxol i.v. 112 ± 12 483 ± 63 371 ± 5862 TxlAn-2 (3:1) i.v. 112 ± 18 236 ± 32 124 ± 14 21 TxlAn-2 (3:1) i.p.105 ± 14 221 ± 50 116 ± 37 19 TxlAn-2 (2:1) i.p. 111 ± 18 215 ± 70 104 ±53 17

These results indicate that the TxlAn conjugates are more potent thanTaxol at inhibiting tumor growth in an in vivo setting. In addition,similar results where obtained whether the conjugate was administeredi.v. or i.p. Finally, similar results where also obtained TxlAnconjugates comprising 2 or 3 Taxol molecules.

Example 17—Effect of Taxol-Angiopep Conjugates Improved Formulation ons.c. Tumor Growth

In a further study, the effect of TxlAn2 (3:1) conjugate formulations ons.c. NCI-H460 or U87 growth was evaluated. Mice were treated by i.p.injections with the improved formulation at 20 mg/kg/day for fiveconsecutive days or by infusion with the implantation of Alzetmini-osmotic pump at a dose of 2 mg/kg/day for 14 days. As shown in FIG.23A and FIG. 23B, the response of mice to TxlAn2 (3:1) conjugateformulation was higher when mice received the improved formulation byinfusion.

These in vivo experiments clearly show the efficacy of the improvedformulation against tumor growth of glioblastoma or lung cancer cells.Similar experiments also indicate the efficacy of these improvedformulations in prolonging survival of animals (data not shown).

The content of each publication, patent and patent application mentionedin the present application is incorporated herein by reference.

Although the present invention has been described in details herein andillustrated in the accompanying drawings, it is to be understood thatthe invention is not limited to the embodiments described herein andthat various changes and modifications may be effected without departingfrom the scope or spirit of the present invention.

The invention claimed is:
 1. A method for the inhibition of cancer cellgrowth, said method comprising administering to a patient having cancera pharmaceutical composition comprising: (a) a conjugate comprising: (i)a polypeptide comprising the amino acid sequence of Angiopep-2; and (ii)an anticancer agent conjugated to said polypeptide; and (b) apharmaceutically acceptable carrier; in an amount sufficient to inhibitcancer cell growth.
 2. The method of claim 1, wherein said cancer is abrain cancer.
 3. The method of claim 2, wherein said brain cancer is abrain metastasis, a glioblastoma, or a glioma.
 4. The method of claim 1,wherein said cancer is outside the brain.
 5. The method of claim 4,wherein said cancer is a lung tumor, a breast tumor, a kidney tumor, aneye tumor, a liver tumor, a colorectal tumor, a tumor of an urinaryorgan, or a metastatic cancer from a breast tumor, a lung tumor, amelanoma, or a brain tumor.
 6. The method of claim 1, wherein saidcancer includes a cell expressing P-glycoprotein.
 7. The method of claim1, wherein said cancer is multiple drug resistant.
 8. The method ofclaim 1, wherein said pharmaceutical composition further comprises: (c)a solubilizer.
 9. The method of claim 8, wherein said solubilizer is apoly-oxyethylene ester of a fatty acid.
 10. The method of claim 9,wherein said poly-oxyethylene ester of a fatty acid is macrogol 15hydroxystearate.
 11. The method of claim 1, wherein said polypeptide isabout 19 amino acids to about 50 amino acids in length.
 12. The methodof claim 1, wherein said polypeptide consists of the amino acid sequenceof Angiopep-2.
 13. The method of claim 1, wherein one to threeanticancer agents are conjugated to said polypeptide.
 14. The method ofclaim 1, wherein said anticancer agent is conjugated to said polypeptideas shown below:

wherein the NH group is either the amino terminal amine or an aminogroup on a lysine at position 10 or position 15 of the Angiopep-2sequence.
 15. The method of claim 1, wherein said anticancer agent ispaclitaxel, vinblastine, vincristine, etoposide, doxorubicin,cyclophosphamide, doclitaxel, melphalan, or chlorambucil.
 16. The methodof claim 15, wherein said anticancer agent is paclitaxel.
 17. The methodof claim 16, wherein said conjugate comprises three molecules ofpaclitaxel.
 18. The method of claim 17, wherein said conjugate has thestructure:


19. The method of claim 1, wherein said pharmaceutical compositionfurther comprises (d) mannitol or (e) a buffer.